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The emerging landscape of non-conventional RNA functions in atherosclerosis

  • Floriana Maria Farina
    Affiliations
    Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximillians-Universität (LMU), Munich, Germany

    German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich, Germany
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  • Christian Weber
    Correspondence
    Corresponding authors. Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximillians-Universität (LMU) of Munich, Pettenkoferstraße 9, 80336, Munich, Germany.
    Affiliations
    Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximillians-Universität (LMU), Munich, Germany

    German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich, Germany

    Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands

    Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
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  • Donato Santovito
    Correspondence
    Corresponding authors. Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximillians-Universität (LMU) of Munich, Pettenkoferstraße 9, 80336, Munich, Germany.
    Affiliations
    Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximillians-Universität (LMU), Munich, Germany

    German Center for Cardiovascular Research (DZHK), Partner Site Munich Heart Alliance, Munich, Germany

    Institute for Genetic and Biomedical Research (IRGB), Unit of Milan, National Research Council, Milan, Italy
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      Highlights

      • Non-coding RNAs (ncRNAs) are a heterogeneous family including miRNAs, tRNAs, snoRNAs, vRNAs, Y RNAs, lncRNAs, and circRNAs.
      • ncRNAs regulate gene expression, translation, ribonucleoprotein localization. Yet, non-conventional functions are emerging.
      • Besides RNA-interference, miRNAs guide epigenetic changes, regulate mitochondrial-encoded genes, affect protein functions.
      • tRNAs and Y RNAs are processed in functionally active oligonucleotides controlling gene expression and RNA-interference.
      • lncRNAs tether intracellular components ruling signaling pathways, and sponge proteins/miRNAs upon processing in circRNAs.

      Abstract

      Most of the human genome is transcribed into non-coding RNAs (ncRNAs), which encompass a heterogeneous family of transcripts including microRNAs (miRNAs), long ncRNAs (lncRNAs), circular RNAs (circRNAs), and others. Although the detailed modes of action of some classes are not fully elucidated, the common notion is that ncRNAs contribute to sculpting gene expression of eukaryotic cells at multiple levels. These range from the regulation of chromatin remodeling and transcriptional activity to post-transcriptional regulation of messenger RNA splicing, stability, and decay. Many of these functions ultimately govern the expression of coding and non-coding genes to affect diverse physiological and pathological mechanisms in vascular biology and beyond. As such, different classes of ncRNAs emerged as crucial regulators of vascular integrity as well as active players in the pathophysiology of atherosclerosis from the early stages of endothelial dysfunction to the clinically relevant complications. However, research in recent years revealed unexpected findings such as small ncRNAs being able to biophysically regulate protein function, the glycosylation of ncRNAs to be exposed on the cell surface, the release of ncRNAs in the extracellular space to act as ligands of receptors, and even the ability of non-coding portion of messenger RNAs to mediate structural functions. This evidence expanded the functional repertoire of ncRNAs far beyond gene regulation and highlighted an additional layer of biological control of cell function. In this Review, we will discuss these emerging aspects of ncRNA biology, highlight the implications for the mechanisms of vascular biology and atherosclerosis, and discuss possible translational implications.

      Graphical abstract

      Keywords

      1. Introduction

      Although its original formulation attempted to address the uncertainty about the flow of information between nucleic acids and proteins, the central dogma of molecular biology has its fundament in the synthesis of messenger RNA (mRNA) from protein-coding genes as an intermediate between DNA and proteins. This led to decades of misperception of RNA as an inert carrier of information (“message”) from DNA to proteins and prompted the belief of RNA as transient and unstructured molecules at the interface between components, owning high-order structures and functional roles. Yet, the role of transfer (tRNAs) and ribosomal RNAs (rRNAs) in protein translation was discovered as early as the 1950s and undoubtedly proved that RNA may assume secondary/ternary structures to exert functions beyond the transfer of information. This discovery inaugurated the non-coding RNA (ncRNA) era. Ignited by the advent of next-generation sequencing, the repertoire of ncRNAs significantly expanded and we are now aware that only a marginal fraction (∼2%) of the mammalian genome is transcribed into “dogmatic” mRNA to be translated into proteins [
      • Palazzo A.F.
      • Lee E.S.
      Non-coding RNA: what is functional and what is junk?.
      ]. Beyond mRNAs and highly abundant rRNAs and tRNAs (accounting for >90% of cellular RNA) [
      • Managadze D.
      • Rogozin I.B.
      • Chernikova D.
      • et al.
      Negative correlation between expression level and evolutionary rate of long intergenic noncoding RNAs.
      ], the human genome is almost entirely transcribed, albeit at low levels, into ncRNAs [
      • Palazzo A.F.
      • Lee E.S.
      Non-coding RNA: what is functional and what is junk?.
      ]. The proportion of the non-coding genome correlates with phylogenetic evolution, and ncRNAs evolutionarily conserved among species show higher expression, possibly implying vital functional roles [
      • Palazzo A.F.
      • Lee E.S.
      Non-coding RNA: what is functional and what is junk?.
      ,
      • Managadze D.
      • Rogozin I.B.
      • Chernikova D.
      • et al.
      Negative correlation between expression level and evolutionary rate of long intergenic noncoding RNAs.
      ]. Nevertheless, the relevance of less abundant ncRNAs is not to be overlooked, as they can act at substoichiometric amounts (e.g., by phase-transition) [
      • Elguindy M.M.
      • Mendell J.T.
      NORAD-induced Pumilio phase separation is required for genome stability.
      ,
      • Unfried J.P.
      • Ulitsky I.
      Substoichiometric action of long noncoding RNAs.
      ], and their expression is finely tuned to enact time- and tissue-specific functions [
      • Mercer T.R.
      • Dinger M.E.
      • Mattick J.S.
      Long non-coding RNAs: insights into functions.
      ].
      Traditionally, ncRNAs are defined by the lack of open reading frames (ORFs) encoding recognizable proteins and are classified by their length into small (smRNAs) and long ncRNAs (lncRNAs) based on a cut-off of 200 nucleotides (nt) [
      • Bartel D.P.
      MicroRNAs: target recognition and regulatory functions.
      ,
      • Mercer T.R.
      • Dinger M.E.
      • Mattick J.S.
      Long non-coding RNAs: insights into functions.
      ]. This classification does not reflect their biochemical roles and researchers have discovered a wide variety of ncRNAs within these two groups. While a consensus on the taxonomy of lncRNAs is still to be reached [
      • Horos R.
      • Buscher M.
      • Kleinendorst R.
      • et al.
      The small non-coding vault RNA1-1 acts as a riboregulator of autophagy.
      ], smRNAs comprise molecules with infrastructural roles (i.e., small nuclear and small nucleolar RNAs) or well-characterized functions (e.g., microRNAs - miRNAs - and tRNAs) (Table 1). Overall, ncRNAs establish a further layer in the epigenetic landscape of mammalian cells by actively shaping the transcriptome at different levels, ranging from transcriptional regulation and chromatin remodeling to splicing and post-transcriptional silencing [
      • Bartel D.P.
      Metazoan MicroRNAs.
      ,
      • Statello L.
      • Guo C.J.
      • Chen L.L.
      • et al.
      Gene regulation by long non-coding RNAs and its biological functions.
      ]. Not surprisingly, research in the last decades unveiled the contribution of multiple classes of ncRNAs to biological processes of crucial physiological relevance, and abnormalities of their expression are inherent to numerous diseases, including cardiovascular disease and atherosclerosis [
      • Chen L.L.
      The expanding regulatory mechanisms and cellular functions of circular RNAs.
      ,
      • Flynn R.A.
      • Pedram K.
      • Malaker S.A.
      • et al.
      Small RNAs are modified with N-glycans and displayed on the surface of living cells.
      ,
      • Boraas L.
      • Hu M.
      • Thornton L.
      • et al.
      Non-coding function for mRNAs in focal adhesion architecture and mechanotransduction.
      ,
      • Santovito D.
      • Weber C.
      Non-canonical features of microRNAs: paradigms emerging from cardiovascular disease.
      ,
      • Peters L.J.F.
      • Biessen E.A.L.
      • Hohl M.
      • et al.
      Small things matter: relevance of MicroRNAs in cardiovascular disease.
      ].
      Table 1Major classes of ncRNAs in mammalian cells.
      SymbolNameSizeCanonical FunctionNon-canonical function
      Small RNAs (smRNAs <200 nt)
      miRNAsmicroRNAs18−24 ntPost-transcriptional mRNA repression.1. Non-canonical MRE targeting

      2. Regulation of gene transcription

      3. Aptamer-like protein regulation
      tRNAsTransfer RNAs76-90 ntIntermediate between mRNA codons and amino acids during translation.1. Regulation of transcriptional fidelity

      2. Cleavage into tRNA fragments (tRFs)
      Y RNAsY-RNAs84-113 ntInvolvement in DNA replication, mRNA fidelity control, and mediating cellular stress.Cleavage into small yRNA fragments (ysRNAs)
      vRNAsVault RNAs88-140 ntIntra- and extracellular trafficking, nucleocytoplasmic shuttling processes, and protein scaffolding1. Protein oligomerization

      2. Impairment of protein recognition
      snoRNAsSmall nucleolar RNAs50-600 ntrRNAs processing in the nucleolus1. Orphan snoRNAs with alternative targeting

      2. Unconventional rRNA modifications

      3. Inhibition of tRFs generation
      Long non-coding RNAs (lncRNAs > 200 nt)
      lncRNAsLong non-coding>200 ntLargely heterogenous functions: mainly involved in cis and trans regulation of gene expression, epigenetic regulation of chromatin accessibility, and regulation of protein function
      circRNAsCircular RNAs>200 ntSponging of miRNAs and RNA-binding proteins with high-affinity binding
      nt, nucleotides.
      Notwithstanding, the last few years shed light on unexpected features that sensibly broadened the functional repertoire of ncRNAs far beyond gene regulation. We now appreciate that ncRNAs directly regulate the function of proteins crucial for cell homeostasis [
      • Horos R.
      • Buscher M.
      • Kleinendorst R.
      • et al.
      The small non-coding vault RNA1-1 acts as a riboregulator of autophagy.
      ,
      • Santovito D.
      • Egea V.
      • Bidzhekov K.
      • et al.
      Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.
      ,
      • Yang D.
      • Wan X.
      • Dennis A.T.
      • et al.
      MicroRNA biophysically modulates cardiac action potential by direct binding to ion channel.
      ], that undergo conformational changes such as circularization [
      • Chen L.L.
      The expanding regulatory mechanisms and cellular functions of circular RNAs.
      ], that acquire atypical localization being exposed on the plasma membrane and released in the extracellular space [
      • Flynn R.A.
      • Pedram K.
      • Malaker S.A.
      • et al.
      Small RNAs are modified with N-glycans and displayed on the surface of living cells.
      ], and that even non-coding portion of mRNA may exert structural functions [
      • Boraas L.
      • Hu M.
      • Thornton L.
      • et al.
      Non-coding function for mRNAs in focal adhesion architecture and mechanotransduction.
      ]. In this Review, we will discuss the emerging aspects of ncRNA biology, highlight their contribution to the mechanisms of atherosclerosis, and discuss future perspectives in this research field with possible therapeutic implications.

      2. microRNAs and their non-canonical functional landscape

      Among metazoan smRNAs, miRNAs are single-stranded sequences ∼22 nt in size that regulate gene expression at the post-transcriptional level to control several biological processes, such as development, differentiation, and homeostasis [
      • Bartel D.P.
      Metazoan MicroRNAs.
      ]. In their canonical biosynthetic pathway (Fig. 1A), miRNAs are transcribed as hairpin-shaped primary transcripts and undergo two maturation steps: the first mediated by DGCR8/DROSHA in the nucleus, and the second by DICER in the cytoplasm [
      • Bartel D.P.
      Metazoan MicroRNAs.
      ]. Variations have been reported [
      • Santovito D.
      • Weber C.
      Non-canonical features of microRNAs: paradigms emerging from cardiovascular disease.
      ], however this pathway culminates in an intermediate miRNA duplex with a strand (guide strand) loaded into Argonaute (AGO) proteins, while the other one (passenger strand) is typically discarded and degraded [
      • Bartel D.P.
      Metazoan MicroRNAs.
      ]. The directionality of the strands determines the name of the mature miRNA: the 5p and the 3p strands originate from the 5′ and 3′end of the precursor miRNA, respectively.
      Fig. 1
      Fig. 1MicroRNA biogenesis and functions.
      (A) Schematics of biogenesis and canonical mode of action of miRNAs. In the nucleus, the RNA polymerase II transcribes pri-miRNAs from mono-/polycistronic MIRNA genes or from introns of protein-coding genes. The microprocessor (DROSHA and DGCR8) processes pri-miRNAs to generate pre-miRNAs that are exported in the cytoplasm by Exportin 5. In the cytoplasm, DICER cleaves pre-miRNAs into ∼20-nt miRNA duplexes. The duplexes are loaded into Argonaute (AGO1-4) proteins with the support of the proteins HSC70 and HSP90. One strand (passenger) is unwound by conformational relaxation of AGO and degraded, while the guide strand is retained for functional pairing. The seed sequence of miRNAs binds to the 3′UTR of target transcripts and guides the assembly of the RISC that contains effector mediators (e.g., the deadenylation complexes CCR4–NOT and PAN2–PAN3) of gene silencing promoting translational repression or decay of target mRNAs. (B) Non-conventional MREs engage in interactions with extra-seed miRNA nucleotides, often by imperfect pairing. MREs located in the CDS induce ribosome stalling and proteasomal degradation of nascent proteins. (C) Nuclear miRNAs bind to promoters and enhancers to regulate gene expression by (1) influencing recruitment of the transcriptional machinery or transcription factors (2) affecting epigenetic enzymes (e.g., EZH2 and MeCP2), to shape chromatin accessibility, (3) mediating RNA-interference in nuclear complexes. (D) AGO2–miRNA complexes can be relocated to mitochondria and alter the expression of mitochondrially encoded genes. (F) Unloaded from AGO proteins, miRNAs can assume aptamer-like secondary structures and bind to functional domains of proteins, regulating their functions. CDS, coding sequence; MRE, miRNA recognition element; ORF, open reading frame; RISC, RNA-induced silencing complex.
      Upon loading into AGO proteins, mature miRNAs recognize their targets by exact Watson-Crick pairing of the seed sequence (nt 2–8 at 5′end of the miRNA) with a complementary miRNA recognition element (MRE) in the 3′-untranslated region (3′UTR) of targeted transcripts [
      • Bartel D.P.
      Metazoan MicroRNAs.
      ]. This step favors the assembly of the RNA-inducing silencing complex (RISC) which contains the effector proteins (e.g., the deadenylation complexes CCR4–NOT and PAN2–PAN3, the decapping DCP1-DCP2 complex, the RNA helicase DDX6) to selectively repress gene expression by blocking the translation or activating nucleolytic degradation of the mRNA (Fig. 1A) [
      • Bartel D.P.
      Metazoan MicroRNAs.
      ]. By this conventional mode of action, miRNAs regulate the expression of genes with profound influence on the processes of atherosclerosis, ranging from endothelial cell (EC) dysfunction to vascular smooth muscle cell (VSMC) plasticity and macrophage activation [
      • Peters L.J.F.
      • Biessen E.A.L.
      • Hohl M.
      • et al.
      Small things matter: relevance of MicroRNAs in cardiovascular disease.
      ,
      • Farina F.M.
      • Hall I.F.
      • Serio S.
      • et al.
      miR-128-3p is a novel regulator of vascular smooth muscle cell phenotypic switch and vascular diseases.
      ,
      • Wei Y.
      • Corbalan-Campos J.
      • Gurung R.
      • et al.
      Dicer in macrophages prevents atherosclerosis by promoting mitochondrial oxidative metabolism.
      ]. However, research in recent years has shown that miRNAs may operate beyond these paradigms. The relevance of these mechanisms for vascular biology and atherosclerosis is emerging and will be discussed in the next paragraphs.

      2.1 Targeting of non-conventional MREs

      The pairing of the seed sequence of the miRNAs with the MRE in the 3′UTR of the target transcript was deemed essential for miRNA-dependent gene silencing and many bioinformatic tools for target prediction rely on this general assumption (Fig. 1B) [
      • Bartel D.P.
      MicroRNAs: target recognition and regulatory functions.
      ]. However, 60% of seed interactions involve non-canonical binding modes with bulged or mismatched nucleotides [
      • Helwak A.
      • Kudla G.
      • Dudnakova T.
      • et al.
      Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding.
      ] and pairing may occur with non-seed nucleotides (e.g., centered pairing) [
      • Shin C.
      • Nam J.W.
      • Farh K.K.
      • et al.
      Expanding the microRNA targeting code: functional sites with centered pairing.
      ], thus extending the miRNA-dependent silencing repertoire beyond the exact complementary seed match. An example of non-canonical interactions relevant to atherosclerosis is provided by the miR-21-dependent repression of Xaf1. Although AGO2-immunoprecipitation revealed the direct targeting, the 3′UTR of Xaf1 does not contain binding sites for miR-21 strands, which rather involves an imperfect centered pairing for miR-21-5p and a 7mer-offset seed matching for miR-21-3p [
      • Schober A.
      • Blay R.M.
      • Saboor Maleki S.
      • et al.
      MicroRNA-21 controls circadian regulation of apoptosis in atherosclerotic lesions.
      ]. The cooperative repression of Xaf1 regulates its circadian expression in lesional macrophages and affects temporal changes in intraplaque apoptosis with imbalanced efferocytosis, leading to a vulnerable plaque phenotype [
      • Schober A.
      • Blay R.M.
      • Saboor Maleki S.
      • et al.
      MicroRNA-21 controls circadian regulation of apoptosis in atherosclerotic lesions.
      ].
      Besides non-canonical pairing modes, unbiased analysis of the human miRNA interactome by CLASH (crosslinking, ligation, and sequencing of hybrids) revealed that only 23.4% of interactions occurred in the 3′UTR, while a high number of hits (42.6%) mapped in the coding sequence (CDS) of mRNAs [
      • Helwak A.
      • Kudla G.
      • Dudnakova T.
      • et al.
      Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding.
      ]. These interactions usually require minimal involvement of the seed sequence but extensive 3′ base-pairing. They promote target repression by ribosome stalling and proteasomal degradation without the need for the classical RISC, which contains adaptor proteins of the TNRC6 family [
      • Helwak A.
      • Kudla G.
      • Dudnakova T.
      • et al.
      Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding.
      ,
      • Zhang K.
      • Zhang X.
      • Cai Z.
      • et al.
      A novel class of microRNA-recognition elements that function only within open reading frames.
      ]. This kind of functional interaction finds its prototype in the repression of DAPK3, which is involved in the regulation of VSMC phenotype [
      • Komatsu S.
      • Ikebe M.
      ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosin II isoforms.
      ], by a member of the vascular-relevant miRNA cluster miR-17-92 [
      • Zhang K.
      • Zhang X.
      • Cai Z.
      • et al.
      A novel class of microRNA-recognition elements that function only within open reading frames.
      ,
      • Chamorro-Jorganes A.
      • Lee M.Y.
      • Araldi E.
      • et al.
      VEGF-induced expression of miR-17-92 cluster in endothelial cells is mediated by ERK/ELK1 activation and regulates angiogenesis.
      ], thus suggesting a possible relevance in vascular biology.
      Finally, while mRNAs are the main binding partners of miRNAs, other classes of RNA have been identified, including ncRNAs. A small proportion of lncRNAs (0.4%) has been detected in CLASH experiments [
      • Helwak A.
      • Kudla G.
      • Dudnakova T.
      • et al.
      Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding.
      ], yet miRNA-mediated repression of lncRNAs importantly affects vascular biology. Indeed, the depletion of mature miRNAs by deletion of Dicer in ECs increased the expression of 97 lncRNAs [
      • Natarelli L.
      • Geissler C.
      • Csaba G.
      • et al.
      miR-103 promotes endothelial maladaptation by targeting lncWDR59.
      ]. Among them, lncWDR39 is a direct target of miR-103-3p and protects against atherosclerosis by maintaining EC proliferative reserve through the Notch1 pathway while preventing the detrimental accrual of DNA damage via coactivation of the β-catenin pathway [
      • Natarelli L.
      • Geissler C.
      • Csaba G.
      • et al.
      miR-103 promotes endothelial maladaptation by targeting lncWDR59.
      ].

      2.2 Transcriptional gene regulation

      The post-transcriptional repression of target transcripts occurs in RISC located in the cytoplasm, yet miRNAs also localize in the nucleus where they may affect gene expression. The nuclear transfer of miRNAs is an active process that involves AGO proteins, with AGO1 possibly favoring nuclear retention [
      • Pitchiaya S.
      • Heinicke L.A.
      • Park J.I.
      • et al.
      Resolving subcellular miRNA trafficking and turnover at single-molecule resolution.
      ]. However, AGO proteins lack nuclear localization signals and additional protein interactors (such as TNRC6A, importin 8, MEX3A) crucially contribute to the shuttling [
      • Santovito D.
      • Egea V.
      • Bidzhekov K.
      • et al.
      Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.
      ,
      • Weinmann L.
      • Hock J.
      • Ivacevic T.
      • et al.
      Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.
      ,
      • Nishi K.
      • Nishi A.
      • Nagasawa T.
      • et al.
      Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus.
      ]. Notably, nuclear localization is fairly specific for some miRNAs, but the detailed mechanisms of specificity are unknown. Specific extra-seed motifs have been associated with nuclear localization, such as the hexanucleotide AGUGUU in the 3′end of miR-29b-3p or the UUGCAUAGU motif for the let-7 family [
      • Hwang H.W.
      • Wentzel E.A.
      • Mendell J.T.
      A hexanucleotide element directs microRNA nuclear import.
      ,
      • Turunen T.A.
      • Roberts T.C.
      • Laitinen P.
      • et al.
      Changes in nuclear and cytoplasmic microRNA distribution in response to hypoxic stress.
      ], but the existence of a unique sequence instructing nuclear enrichment is still debated and further studies are required to clarify the mechanisms of nuclear shuttling.
      In the nucleus, miRNAs may bind to promoters to enhance or repress gene transcription (Fig. 1C). While the exact mechanisms are not fully elucidated, most of the studies highlight the requirement of AGO proteins and the possible contribution of promoter RNAs, which may facilitate changes in chromatin conformation to favor binding to DNA [
      • Li H.
      • Zhan J.
      • Zhao Y.
      • et al.
      Identification of ncRNA-mediated functions of nucleus-localized miR-320 in cardiomyocytes.
      ]. Activation of the transcription may occur when miRNAs bind the TATA-box motifs and promote the AGO1-dependent recruitment of a pre-initiation transcription complex made of RNA polymerase II and TATA-binding protein (TBP). This is exemplified by let-7i which enhances the transcription of IL2 in CD4+ T-cells [
      • Zhang Y.
      • Fan M.
      • Zhang X.
      • et al.
      Cellular microRNAs up-regulate transcription via interaction with promoter TATA-box motifs.
      ]. Otherwise, miRNAs activate transcription by direct binding with gene enhancers. In this case, the miRNA (e.g., miR-24) engages with its seed sequence in AGO2-dependent interactions with the enhancer and affects chromatin accessibility (i.e., higher H3K27ac and lower H3K9me3), ultimately leading to the recruitment of the RNA-polymerase II and the transcriptional co-activator proteins p300/CBP [
      • Xiao M.
      • Li J.
      • Li W.
      • et al.
      MicroRNAs activate gene transcription epigenetically as an enhancer trigger.
      ]. Over 300 miRNA loci map in genomic areas in the proximity of active enhancers, thus suggesting the possible wide relevance of this mode of action.
      On the other hand, nuclear miRNAs may repress transcription by diverse mechanisms. Indeed, miRNAs may pair DNA-binding motifs for transcription factors and allosterically prevent their interaction and the recruitment of the transcription machinery. This is epitomized by the ability of miR-30b-5p to bind specific palindromic motifs in the promoter of lysosome- and autophagy-related genes and inhibit the interaction with the transcription factor TFEB, with profound effects on the activation of autophagy [
      • Guo H.
      • Pu M.
      • Tai Y.
      • et al.
      Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy.
      ]. Alternatively, miRNAs may contribute to the recruitment of epigenetic enzymes that regulate chromatin accessibility. For instance, miR-320 recruits the enzyme Enhancer of Zeste Homolog 2 (EZH2) to the gene promoter of POLR3D through an indirect interaction mediated by AGO1 [
      • Kim D.H.
      • Saetrom P.
      • Snove Jr., O.
      • et al.
      MicroRNA-directed transcriptional gene silencing in mammalian cells.
      ]. EZH2 is a part of the polycomb repressive complex 2 (PRC2) and catalyzes the trimethylation of histone 3 at Lys27 (H3K27me3), thus cis-repressing POLR3D transcription [
      • Kim D.H.
      • Saetrom P.
      • Snove Jr., O.
      • et al.
      MicroRNA-directed transcriptional gene silencing in mammalian cells.
      ]. Notably, EZH2 is also involved in the repressive function of nuclear let-7d. In this case, the miRNA forms an RNA-duplex by binding nuclear ncRNAs bidirectionally transcribed with the target genes. This duplex allows the assembly of a complex (named MiCEE) composed of the proteins C1D, EXOSC10, and EZH2. This complex eventually degrades the let-7d-bound ncRNAs, represses the transcription of the target genes through EZH2, and tethers the regulated loci to the nucleolus [
      • Singh I.
      • Contreras A.
      • Cordero J.
      • et al.
      MiCEE is a ncRNA-protein complex that mediates epigenetic silencing and nucleolar organization.
      ]. Another epigenetic enzyme, the methylation reader MeCP2, has been shown to bind nuclear miRNAs (i.e., let-7i, miR-126, miR-375) which guide its localization toward specific chromatin areas to repress gene transcription [
      • Khan A.W.
      • Ziemann M.
      • Rafehi H.
      • et al.
      MeCP2 interacts with chromosomal microRNAs in brain.
      ]. Finally, miRNAs can exert RNA-interference in the nucleus where the required components can assemble into multimolecular complexes [
      • Gagnon K.T.
      • Li L.
      • Chu Y.
      • et al.
      RNAi factors are present and active in human cell nuclei.
      ]. Nuclear complexes differ from cytoplasmic RISCs in size and composition but are incapable of AGO loading, thus suggesting that AGO-miRNA complexes form in the cytoplasm before nuclear shuttling [
      • Pitchiaya S.
      • Heinicke L.A.
      • Park J.I.
      • et al.
      Resolving subcellular miRNA trafficking and turnover at single-molecule resolution.
      ,
      • Nishi K.
      • Nishi A.
      • Nagasawa T.
      • et al.
      Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus.
      ]. Still, mechanisms of target identification appear similar and nuclear miRNA-interference extends the silencing repertoire to introns and other nuclear ncRNAs [
      • Weinmann L.
      • Hock J.
      • Ivacevic T.
      • et al.
      Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.
      ,
      • Sarshad A.A.
      • Juan A.H.
      • Muler A.I.C.
      • et al.
      Argonaute-miRNA complexes silence target mRNAs in the nucleus of mammalian stem cells.
      ].
      A systematic scrutiny of the role of miRNA-directed transcriptional regulation in vascular biology is lacking. Yet, validated targets of nuclear miRNAs are relevant contributors to mechanisms of atherogenesis. For example, nuclear miR-320 regulates the transcription of the scavenger receptor CD36 affecting the uptake of free fatty acids and the accumulation of lipotoxic diacylglycerol [
      • Li H.
      • Zhan J.
      • Zhao Y.
      • et al.
      Identification of ncRNA-mediated functions of nucleus-localized miR-320 in cardiomyocytes.
      ,
      • Li H.
      • Fan J.
      • Zhao Y.
      • et al.
      Nuclear miR-320 mediates diabetes-induced cardiac dysfunction by activating transcription of fatty acid metabolic genes to cause lipotoxicity in the heart.
      ]. Moreover, nuclear miR-30b-5p dampens the transcription of autophagy-related genes involved in the initiation and elongation of autophagy, a stress-response process with crucial implications for atherogenesis [
      • Guo H.
      • Pu M.
      • Tai Y.
      • et al.
      Nuclear miR-30b-5p suppresses TFEB-mediated lysosomal biogenesis and autophagy.
      ,
      • Henderson J.M.
      • Weber C.
      • Santovito D.
      Beyond self-recycling: cell-specific role of autophagy in atherosclerosis.
      ]. Finally, nuclear miR-9-5p silence by AGO2-dependent RNA-interference the lncRNA Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), which plays a protective role against atherogenesis [
      • Leucci E.
      • Patella F.
      • Waage J.
      • et al.
      microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus.
      ,
      • Cremer S.
      • Michalik K.M.
      • Fischer A.
      • et al.
      Hematopoietic deficiency of the long noncoding RNA MALAT1 promotes atherosclerosis and plaque inflammation.
      ].

      2.3 Regulation of mitochondrial genes

      Although mitochondria lack components for endogenous miRNA biogenesis, they import miRNAs associated with AGO2 from the hosting cell (Fig. 1D) in a process that may involve the protein polynucleotide phosphorylase [
      • Shepherd D.L.
      • Hathaway Q.A.
      • Pinti M.V.
      • et al.
      Exploring the mitochondrial microRNA import pathway through Polynucleotide Phosphorylase (PNPase).
      ]. None of the RISC components is found in mitochondria besides AGO2, nonetheless miRNAs control the expression of mitochondrial genes and influence oxidative phosphorylation and reactive oxygen species (ROS) production. The evidence on miRNA-dependent regulation of mitochondrial genes derives from studies on myocytes [
      • Zhang X.
      • Zuo X.
      • Yang B.
      • et al.
      MicroRNA directly enhances mitochondrial translation during muscle differentiation.
      ,
      • Li H.
      • Zhang X.
      • Wang F.
      • et al.
      MicroRNA-21 lowers blood pressure in spontaneous hypertensive rats by upregulating mitochondrial translation.
      ], but some miRNAs are well expressed in vascular cells possibly translating mechanisms to vascular biology. Among them, miR-21-5p, miR-92a-5p, and let-7b-5p reduce ROS generation by enhancing the expression of the mitochondrially-encoded cytochrome b (mt-Cytb), a mandatory protein for the function of complex III in the respiratory chain [
      • Li H.
      • Zhang X.
      • Wang F.
      • et al.
      MicroRNA-21 lowers blood pressure in spontaneous hypertensive rats by upregulating mitochondrial translation.
      ,
      • Li H.
      • Dai B.
      • Fan J.
      • et al.
      The different roles of miRNA-92a-2-5p and let-7b-5p in mitochondrial translation in db/db mice.
      ], while miR-146 attenuates mitochondrial dysfunction and prevent apoptosis by targeting cyclophilin D [
      • Su Q.
      • Xu Y.
      • Cai R.
      • et al.
      miR-146a inhibits mitochondrial dysfunction and myocardial infarction by targeting cyclophilin D.
      ]. As miRNAs can exert distinct and possibly conflictual functions in cytoplasmic RISCs and mitochondria (as for miR-92a-5p and let-7b-5p) [
      • Li H.
      • Dai B.
      • Fan J.
      • et al.
      The different roles of miRNA-92a-2-5p and let-7b-5p in mitochondrial translation in db/db mice.
      ], further studies are entailed to understand the mechanisms with regard to possible therapeutic applications.

      2.4 Aptamer-like protein interactions

      The common features of the modes of action described so far are the loading into AGO proteins and the interaction with nucleic acids, either RNA or DNA. However, stoichiometric analyses revealed a 13-fold excess of miRNAs relative to AGO proteins in human cells [
      • Janas M.M.
      • Wang B.
      • Harris A.S.
      • et al.
      Alternative RISC assembly: binding and repression of microRNA-mRNA duplexes by human Ago proteins.
      ]. This observation suggests that a large portion of miRNAs is stably retained outside the RISC and available for molecular interactions. Notably, mature miRNAs may assume secondary structures in solution resembling aptamers and recent studies clearly showed that some highly expressed miRNAs (miR-126-5p and miR-1-3p) engage in biophysical interaction with proteins to regulate their function (Fig. 1E) [
      • Santovito D.
      • Egea V.
      • Bidzhekov K.
      • et al.
      Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.
      ,
      • Yang D.
      • Wan X.
      • Dennis A.T.
      • et al.
      MicroRNA biophysically modulates cardiac action potential by direct binding to ion channel.
      ,
      • Belter A.
      • Gudanis D.
      • Rolle K.
      • et al.
      Mature miRNAs form secondary structure, which suggests their function beyond RISC.
      ].
      The first report of an aptamer-like function of a miRNA is the biophysical interaction between the miR-126-5p and the effector caspase 3 and holds relevance for the maintenance of endothelial integrity. This interaction occurs in ECs upon activation of autophagy (by rapamycin or high-shear stress) which induces nuclear shuttling of miR-126-5p in a complex with AGO2 and MEX3A. In the nucleus, miR-126-5p dissociates from AGO2 and MEX3A to interact with the catalytic pocket and heterodimer interface of caspase 3, thus inhibiting its proteolytic activity and blocking apoptosis in vivo [
      • Santovito D.
      • Egea V.
      • Bidzhekov K.
      • et al.
      Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.
      ]. Complete clarification of the structural bases of this interaction requires further investigations, as well as the role of MEX3A, which facilitates the interaction with caspase 3 despite not being involved in the inhibitory function of miR-126-5p. Nonetheless, the biophysical inhibition of a protease by a miRNA represents a significant paradigm shift in miRNA biology.

      3. Non-structural roles of transfer RNAs

      Transfer RNAs (tRNAs) are key components of the eukaryotic translation machinery and are among the most extensively studied RNA molecules. They lay at the interface between mRNAs and proteins as they provide the corresponding amino acid upon pairing with specific codons in the mRNA, thus settling the elongation of nascent polypeptides (Fig. 2A) [
      • Hoagland M.B.
      • Stephenson M.L.
      • Scott J.F.
      • et al.
      A soluble ribonucleic acid intermediate in protein synthesis.
      ]. Generally, tRNAs hold a distinctive cloverleaf secondary structure, characterized by the two arms (D and T), an acceptor stem, and an anticodon stem loop (Fig. 2B). They usually assume a three-dimensional L-shaped structure, ensuring the formation of base pairs in between nucleotides in several parts of the molecule (Fig. 2C) [
      • Kim S.H.
      • Quigley G.J.
      • Suddath F.L.
      • et al.
      Three-dimensional structure of yeast phenylalanine transfer RNA: folding of the polynucleotide chain.
      ].
      Fig. 2
      Fig. 2Structural and functional features of transfer RNAs (tRNAs).
      (A) The distinctive secondary structure of tRNAs resembling a cloverleaf with two arms (D and T), an acceptor (A) stem, a variable (V) loop, and an anticodon (AC) stem-loop. (B) Canonical function of tRNAs in supplying the amino acid corresponding codon in the mRNA sequence. (C) Conformational switch from a L-shaped (top panel) to a λ-shaped (bottom panel) three-dimensional structure allows binding to snoRNAs in the nucleolus. For each conformation, schematic and crystallography are presented (PDB code: 1J2B). (C) Small tRNA-derived fragments (tRFs) originate from tRNAs and regulates gene transcription and RNA-interference.
      Although the knowledge about tRNAs was certain to have reached its entirety decades ago, new roles of these molecules in various processes have been demonstrated. For example, the enzyme ArcTGT determines rearrangements of the L-shaped structure of tRNAs toward a non-canonical three-dimensional conformation termed λ-shape (Fig. 2C). Upon conformational change, tRNAs can bind snoRNAs in the nucleolus and control the synthesis of snRNAs and rRNAs [
      • Ishitani R.
      • Nureki O.
      • Nameki N.
      • et al.
      Alternative tertiary structure of tRNA for recognition by a posttranscriptional modification enzyme.
      ]. Moreover, variations of tRNA nucleotides, especially when occurring at the anticodon loop, might affect translation fidelity [
      • Motorin Y.
      • Helm M.
      tRNA stabilization by modified nucleotides.
      ]. This phenomenon is called “wobble” coupling and is related to non-canonical pairing between nucleotides (e.g., G with a C or U, but not with A), to guarantee the maintenance of reading fidelity [
      • Kothe U.
      • Rodnina M.V.
      Codon reading by tRNAAla with modified uridine in the wobble position.
      ]. Finally, some aminoacyl-tRNA synthetases (e.g., p-cyanophenylalanine) exhibit polyspecificity for amino acids not normally available in nature, while being able to discriminate against the classical 20 amino acids [
      • Young D.D.
      • Young T.S.
      • Jahnz M.
      • et al.
      An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity.
      ]. Little is known about the functional relevance, but recent evidence raises the hypothesis of a mechanism to prevent nonsense suppression due to premature unintended stop codons (Fig. 2D) [
      • Albers S.
      • Beckert B.
      • Matthies M.C.
      • et al.
      Repurposing tRNAs for nonsense suppression.
      ].
      Besides conformational changes and non-canonical functions to ensure translational reliability, tRNAs can undergo cleavage to produce a class of smRNAs termed tRNA-derived fragments (tRFs) (Fig. 2D) [
      • Fu H.
      • Feng J.
      • Liu Q.
      • et al.
      Stress induces tRNA cleavage by angiogenin in mammalian cells.
      ]. Contrary to tRNA-like structures (TLSs), which possess tertiary structures similar to tRNAs, small tRFs (<32 nt) are stochastically produced by DICER and DROSHA, although alternative biosynthetic pathways have been proposed (involving angiogenin or other exonucleases) [
      • Li Z.
      • Ender C.
      • Meister G.
      • et al.
      Extensive terminal and asymmetric processing of small RNAs from rRNAs, snoRNAs, snRNAs, and tRNAs.
      ]. The synthesis of tRFs is induced by stress conditions, such as hypoxia [
      • Fu H.
      • Feng J.
      • Liu Q.
      • et al.
      Stress induces tRNA cleavage by angiogenin in mammalian cells.
      ], and many studies indicate that tRFs abundance does not reflect the expression of their derivative tRNAs [
      • Krishna S.
      • Yim D.G.
      • Lakshmanan V.
      • et al.
      Dynamic expression of tRNA-derived small RNAs define cellular states.
      ]. Notably, recent evidence revealed the regulation of multiple tRFs in VSMCs acquiring a proliferative phenotype. Among them, AS-tDR-000067 plays a mechanistic role by binding the promoter of the TP53 gene to suppress its transcription, hence promoting cell proliferation with possible implications for vascular remodeling (Fig. 2D), as proliferative VSMCs contribute to atherogenesis by neointima formation [
      • Zhao J.Z.
      • Li Q.Y.
      • Lin J.J.
      • et al.
      Integrated analysis of tRNA-derived small RNAs in proliferative human aortic smooth muscle cells.
      ]. Finally, due to their similarity with miRNAs and siRNAs, extensive emphasis has been given to the possible involvement of tRFs in the mechanisms of RNA-interference. Studies in recent years showed the ability of rRFs to bind AGO proteins and guide the cytoplasmic RISC toward target transcripts [
      • Kumar P.
      • Anaya J.
      • Mudunuri S.B.
      • et al.
      Meta-analysis of tRNA derived RNA fragments reveals that they are evolutionarily conserved and associate with AGO proteins to recognize specific RNA targets.
      ,
      • Kuscu C.
      • Kumar P.
      • Kiran M.
      • et al.
      tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner.
      ], or to mediate the silencing of nascent RNAs in the nucleus [
      • Di Fazio A.
      • Schlackow M.
      • Pong S.K.
      • et al.
      Dicer dependent tRNA derived small RNAs promote nascent RNA silencing.
      ]. In the example, tRF1s target the 3′UTR of Timp3 with repercussions in cardiac remodeling and fibrosis, while tRF3s mediate RNA-silencing of PCSK9, a crucial negative regulator of LDL-receptor mechanistically implied in dyslipidemia and atherogenesis [
      • Shen L.
      • Gan M.
      • Tan Z.
      • et al.
      A novel class of tRNA-derived small non-coding RNAs respond to myocardial hypertrophy and contribute to intergenerational inheritance.
      ,
      • Green J.A.
      • Ansari M.Y.
      • Ball H.C.
      • et al.
      tRNA-derived fragments (tRFs) regulate post-transcriptional gene expression via AGO-dependent mechanism in IL-1beta stimulated chondrocytes.
      ].
      In line with their involvement in atherosclerosis, deep RNA sequencing of human atherosclerotic plaques unveiled distinct tRFs expression patterns compared to healthy arteries. In particular, tRF2 and tRF3s were reduced in atherosclerotic plaques, where the predominant tRF subtype was the tRF5s [
      • He X.
      • Yang Y.
      • Wang Q.
      • et al.
      Expression profiles and potential roles of transfer RNA-derived small RNAs in atherosclerosis.
      ,
      • Wang J.
      • Dong P.K.
      • Xu X.F.
      • et al.
      Identification of tRNA-derived fragments and their potential roles in atherosclerosis.
      ]. Within this subtype, tRF-Gly-GCC-004 regulates the expression of genes involved in cell adhesion (e.g., ICAM1) and proliferation (e.g., CDK1, MYC, KIF20A, OLR1), conceivably by RNA-interference mechanisms [
      • He X.
      • Yang Y.
      • Wang Q.
      • et al.
      Expression profiles and potential roles of transfer RNA-derived small RNAs in atherosclerosis.
      ]. Consistently, tRF-Gly-GCC-004 overexpression in vitro enhanced monocyte adhesion to ECs and promoted a proliferative phenotype of VSMCs [
      • He X.
      • Yang Y.
      • Wang Q.
      • et al.
      Expression profiles and potential roles of transfer RNA-derived small RNAs in atherosclerosis.
      ]. Finally, tRF-Gly-GCC-009 is also upregulated in human atherosclerosis and in silico analysis suggests a regulatory role of genes entailed in cell adhesion, as well as in the apelin and Notch pathways, although experimental validation is still lacking [
      • Wang J.
      • Dong P.K.
      • Xu X.F.
      • et al.
      Identification of tRNA-derived fragments and their potential roles in atherosclerosis.
      ]. Together, these findings indicate the relevance of tRFs in atherosclerosis, and future studies are warranted to clarify mechanisms and potential therapeutic benefits of their manipulation.

      4. Small nucleolar RNAs (snoRNAs): atypical targets and non-canonical functions

      Identified in archaea and eukaryotes, snoRNAs are one of the oldest evolutionary conserved RNA classes, and their abundance correlates with the organism complexity (from 76 in Saccharomyces cerevisiae to 1000 in humans) [
      • Omer A.D.
      • Lowe T.M.
      • Russell A.G.
      • et al.
      Homologs of small nucleolar RNAs in Archaea.
      ]. They are abundantly expressed smRNAs (50–500 nt) critically engaged in post-transcriptional rRNA processing [
      • Lange T.S.
      • Ezrokhi M.
      • Amaldi F.
      • et al.
      Box H and box ACA are nucleolar localization elements of U17 small nucleolar RNA.
      ]. In humans, RNA polymerase II transcribes snoRNAs principally from independent genes. However, snoRNAs may derive from the splicing of polycistronic transcripts encoded within introns of coding and non-coding genes, especially in other species (Fig. 3A). In eukaryotic cells, snoRNAs reside in distinct topological foci of the nucleolus, which is the most important nuclear substructure and the site of rRNA transcription, maturation, and ribosome assembly. They are characterized by the presence of two distinct sets of motifs, either C/D or H/ACA boxes, and up to two antisense sequences pairing target RNAs (Fig. 3B). The canonical function of snoRNAs is to guide epitranscriptomic rRNA modifications. To this end, the antisense motifs engage in Watson-Crick base pairing with target rRNAs, while the C/D or H/ACA boxes act as scaffolds for effector proteins. Specifically, C/D-box snoRNAs are associated with four evolutionarily conserved proteins, namely Fibrillarin (FBL), SNU13, NOP58, NOP56, and drive 2′-O-ribose methylation (2′Ome). On the other hand, H/ACA-box snoRNAs operate as scaffolds for four other proteins named NHP2, NOP10, GAR1, and dyskerin (DKC1), the enzyme catalyzing RNA pseudo-uridylation (Ψ) (Fig. 3B) [
      • Watkins N.J.
      • Bohnsack M.T.
      ].
      Fig. 3
      Fig. 3Biogenesis and functions of small-nucleolar RNAs (snoRNAs).
      (A) Schematics of snoRNA biogenesis. In the nucleus, RNA polymerase II transcribes snoRNAs from introns of coding/non-coding genes or from independent genes. snoRNAs are transferred to the cytoplasm and are characterized by sequence motifs, either C/D or H/ACA boxes, that define two major functional categories. In the cytoplasm, snoRNAs may be cleaved into smaller oligonucleotides termed sdRNAs, possibly by Dicer. (B) Canonical function of snoRNAs as protein scaffold to protein to promote 2′O-Methylation (C/D box snoRNAs) or 5′pseudouridynilation (H/ACA box snoRNAs). (C) Examples of non-canonical snoRNAs function: orphan snoRNAs targeting RNA species other than rRNAs, prevention of tRNA cleavage, repurposing epigenetic enzymes to promote additional rRNA modifications, and cleavage of snoRNAs to generate sdRNAs involved in RNA-interference, nuclear exosome complex, and pre-mRNA stabilization.
      Besides these conventional epitrascriptomic modifications of rRNAs, atypical targets and non-canonical functions of snoRNAs emerged (Fig. 3C). Notably, the antisense sequences of many C/D-box snoRNA do not match with any known rRNA 2′Ome sites. These snoRNAs (e.g., SNORA73) are considered orphans and plausibly engage in alternative targeting to direct 2′Ome modifications toward other RNA species, including mRNAs and tRNAs [
      • Elliott B.A.
      • Ho H.T.
      • Ranganathan S.V.
      • et al.
      Modification of messenger RNA by 2'-O-methylation regulates gene expression in vivo.
      ]. The biological relevance of these modifications is reflected by changes in the stability and translation of mRNAs, while 2′OMe modifications of tRNAs affect the site-specific generation of tRFs, without influencing mature tRNAs stability [
      • Elliott B.A.
      • Ho H.T.
      • Ranganathan S.V.
      • et al.
      Modification of messenger RNA by 2'-O-methylation regulates gene expression in vivo.
      ,
      • van Ingen E.
      • Engbers P.A.M.
      • Woudenberg T.
      • et al.
      C/D box snoRNA SNORD113-6 guides 2'-O-methylation and protects against site-specific fragmentation of tRNA(Leu)(TAA) in vascular remodeling.
      ]. Strikingly, orphan C/D-box snoRNAs are also endowed with functions completely diverging from 2′Ome modifications. Among them, the snoRNA U13 guides rRNA acetylation by redirecting the activity of the enzyme NAT10, an acetyltransferase mostly targeting histones and microtubules [
      • Sharma S.
      • Langhendries J.L.
      • Watzinger P.
      • et al.
      Yeast Kre33 and human NAT10 are conserved 18S rRNA cytosine acetyltransferases that modify tRNAs assisted by the adaptor Tan1/THUMPD1.
      ]. Besides NAT10, snoRNAs instruct other epigenetic enzymes (i.e., EZH2) to enact functions independently from canonical histone modifications, including rRNA methylation to control translational initiation [
      • Yi Y.
      • Li Y.
      • Meng Q.
      • et al.
      A PRC2-independent function for EZH2 in regulating rRNA 2'-O methylation and IRES-dependent translation.
      ]. Moreover, snoRNAs contribute to regulating alternative splicing and processing of 3′UTRs by diverting polyadenylation sites, can compete for functional binding sites of RNA-binding proteins, and even sequester specific proteins [
      • van Ingen E.
      • Engbers P.A.M.
      • Woudenberg T.
      • et al.
      C/D box snoRNA SNORD113-6 guides 2'-O-methylation and protects against site-specific fragmentation of tRNA(Leu)(TAA) in vascular remodeling.
      ]. Finally, some C/D-box snoRNAs are cleaved (possibly by DICER) into smaller RNA fragments (termed sdRNAs), which may be loaded into AGO proteins to inhibit mRNA translation like miRNAs, control the nuclear exosome complex, and stabilize pre-mRNAs [
      • Brameier M.
      • Herwig A.
      • Reinhardt R.
      • et al.
      Human box C/D snoRNAs with miRNA like functions: expanding the range of regulatory RNAs.
      ,
      • Ender C.
      • Krek A.
      • Friedlander M.R.
      • et al.
      A human snoRNA with microRNA-like functions.
      ,
      • Gudipati R.K.
      • Xu Z.
      • Lebreton A.
      • et al.
      Extensive degradation of RNA precursors by the exosome in wild-type cells.
      ,
      • Han C.
      • Sun L.Y.
      • Luo X.Q.
      • et al.
      Chromatin-associated orphan snoRNA regulates DNA damage-mediated differentiation via a non-canonical complex.
      ].
      Pioneering work on the H/ACA-box snoRNA U17 revealed its important contribution to cellular cholesterol trafficking. U17 negatively regulates the expression of the mitochondrial adaptor protein HUMMR, thus acting as a switch controlling cholesterol flux from the plasma membrane toward the mitochondria or the endoplasmatic reticulum. This ultimately affects the cholesterol pool accessible to ACAT for esterification, with implications for oxysterol synthesis and cellular lipid storage [
      • Jinn S.
      • Brandis K.A.
      • Ren A.
      • et al.
      snoRNA U17 regulates cellular cholesterol trafficking.
      ]. Notably, the HUMMR transcript lacks motifs complementary to U17 antisense sequences and was not pseudo-uridylated, implying mechanisms independent of canonical pseudo-uridylation [
      • Jinn S.
      • Brandis K.A.
      • Ren A.
      • et al.
      snoRNA U17 regulates cellular cholesterol trafficking.
      ]. However, the role of snoRNAs in cell metabolism extends well beyond cholesterol trafficking. For instance, SNORNA73 masters a broad rewiring of the oxidative metabolism in crosstalk with the mTOR pathway to preserve cellular rRNA content by controlling pre-rRNA cleavage, without involving pseudo-uridylation [
      • Sletten A.C.
      • Davidson J.W.
      • Yagabasan B.
      • et al.
      Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis.
      ]. Notably, the silencing of Snorna73 protects against the development of hepatic steatosis in vivo [
      • Sletten A.C.
      • Davidson J.W.
      • Yagabasan B.
      • et al.
      Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis.
      ]. Finally, non-canonical functions of snoRNAs contribute to the pathophysiology of β-cells. In particular, the deletion of the four C/D-box snoRNAs encoded in the Rpl13a locus (i.e., U32a, U33, U34, U35) increased the glucose-triggered secretion of insulin, improving glucose tolerance and reducing the detrimental effects of diabetogenic stimuli [
      • Lee J.
      • Harris A.N.
      • Holley C.L.
      • et al.
      Rpl13a small nucleolar RNAs regulate systemic glucose metabolism.
      ]. Together, this evidence reveals the prominence of non-canonical functions of snoRNAs in the comprehensive regulation of metabolism and highlights their therapeutic opportunity to treat metabolic diseases, which are crucial risk factors for atherogenesis.
      The involvement of non-canonical features of snoRNAs in vascular biology surfaced in recent years. The snoRNA SNORD113-6 (human homolog of murine Af357435) regulates the expression of multiple genes in the integrin pathway by affecting stability, processing, and splicing of pre-mRNAs through 2′Ome modifications [
      • van Ingen E.
      • van den Homberg D.A.L.
      • van der Bent M.L.
      • et al.
      C/D box snoRNA SNORD113-6/AF357425 plays a dual role in integrin signalling and arterial fibroblast function via pre-mRNA processing and 2'O-ribose methylation.
      ]. Moreover, SNORD113-6 binds to tRNAs (e.g., tRNALeu) and competes with tRNAs endonucleases preventing the formation of tRFs in adventitial fibroblasts [
      • van Ingen E.
      • Engbers P.A.M.
      • Woudenberg T.
      • et al.
      C/D box snoRNA SNORD113-6 guides 2'-O-methylation and protects against site-specific fragmentation of tRNA(Leu)(TAA) in vascular remodeling.
      ], delineating an intriguing crosstalk of non-canonical functions of diverse ncRNAs (Fig. 3C). SNORD113-6 is encoded in the 14q32 locus and highly expressed in human vessels and primary ECs and VSMCs [
      • Hakansson K.E.J.
      • Goossens E.A.C.
      • Trompet S.
      • et al.
      Genetic associations and regulation of expression indicate an independent role for 14q32 snoRNAs in human cardiovascular disease.
      ]. Silencing of SNORD113-6 in vascular fibroblasts in vitro affected cell migration, proliferation, contraction, and cell-matrix and cell-cell interactions, implying a possible influence on vascular function [
      • van Ingen E.
      • van den Homberg D.A.L.
      • van der Bent M.L.
      • et al.
      C/D box snoRNA SNORD113-6/AF357425 plays a dual role in integrin signalling and arterial fibroblast function via pre-mRNA processing and 2'O-ribose methylation.
      ]. Consistent with a translational perspective, SNORD113-6 expression is upregulated in patients with cardiovascular disease (i.e., end-stage heart failure) [
      • van Ingen E.
      • van den Homberg D.A.L.
      • van der Bent M.L.
      • et al.
      C/D box snoRNA SNORD113-6/AF357425 plays a dual role in integrin signalling and arterial fibroblast function via pre-mRNA processing and 2'O-ribose methylation.
      ]. Other snoRNAs in the 14q32 locus, namely SNORD113-2 and SNORD114-1, were implicated in vascular disease as their circulating plasma levels were higher in patients with peripheral artery disease and showed signals of association with platelet reactivity [
      • Nossent A.Y.
      • Ektefaie N.
      • Wojta J.
      • et al.
      Plasma levels of snoRNAs are associated with platelet activation in patients with peripheral artery disease.
      ]. Yet, the functional role of snoRNAs in vascular biology requires further investigation and may provide insights into the therapeutic opportunity to switch chemical RNA modifications and regulate pathways relevant to vascular disease.

      5. Emerging classes of small non-coding RNAs

      5.1 Vault RNAs

      Around the mid-1980s, a novel category of ribosomal structures was identified from ovoid bodies within rat liver and named “vault RNAs” (vRNAs), owning their shape resembling cathedral vaults [
      • Kedersha N.L.
      • Rome L.H.
      Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA.
      ]. This peculiar structure is contributed by the interaction with three different proteins: the major vault protein (MVP), the vault poly-ADP-ribose polymerase (VPARP), and the telomerase-associated protein-1 (TEP-1) (Fig. 4A) [
      • Kedersha N.L.
      • Heuser J.E.
      • Chugani D.C.
      • et al.
      Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry.
      ,
      • Lee K.
      • Kunkeaw N.
      • Jeon S.H.
      • et al.
      Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity.
      ]. The vRNA complex is in a hollow and barrel‐shaped ribonucleoprotein (RNP) with a large internal volume to encapsulate a great number of proteins, mediating intra- and extracellular transport. Humans express four vRNAs, which are 88–140 nt long and transcribed by RNA polymerase III from two loci on chromosome 5q31: VTRNA-1 encoding for vtRNA1-1, vtRNA1-2, vtRNA1-3; and VTRNA-2 for vtRNA2-1 (also known as pre-miR-886) [
      • Lee K.
      • Kunkeaw N.
      • Jeon S.H.
      • et al.
      Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity.
      ,
      • Kickhoefer V.A.
      • Searles R.P.
      • Kedersha N.L.
      • et al.
      Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III.
      ]. Their expression is stimulated by the transcription factors CREB and AP-1, which are involved in the transcriptional response to cellular stress. For this reason, several pathological conditions increase vRNA expression that, due to their short half-life (∼1 h), may act as signalers of altered cellular states. Intriguingly, the expression of vRNAs is governed by epigenetic modifications, such as changes in DNA methylation in the VTRNA-2 promoter or in CpG accessibility of distant regulatory elements of the VTRNA-1 promoter, possibly implying cell-specific transcriptional regulation of the three vRNAs [
      • Helbo A.S.
      • Lay F.D.
      • Jones P.A.
      • et al.
      Nucleosome positioning and NDR structure at RNA polymerase III promoters.
      ,
      • Helbo A.S.
      • Treppendahl M.
      • Aslan D.
      • et al.
      Hypermethylation of the VTRNA1-3 promoter is associated with poor outcome in lower risk myelodysplastic syndrome patients.
      ].
      Fig. 4
      Fig. 4Emerging non-coding RNA species: vault RNAs (vRNAs) and Y RNAs.
      (A) Schematics of biogenesis and secondary structure of human vRNAs and the protein included in the vault complex. The characteristic shape of the complex is determined by the interaction of vRNAs with the major vault protein (MVP), the vault poly-ADP-ribose polymerase (VPARP), and the telomerase-associated protein-1 (TEP-1), necessary for vRNA stabilization. For each protein schematic and crystallography are presented (PDB codes: 4V60, 4HL8, 4LNV). (B) Structural schematics of vault complex, subcellular localization, and canonical function. (C) Exemplary non-canonical functions: direct mechanism of action of vtRNA1-1 which binds the ZZ-domains of p62/SQSTM1 and prevents its oligomerization to block the autophagic flux. Indirect function of vRNA mediated by MVP which prevents TRAF6 ubiquitination and dampens the proinflammatory NF-κB pathway. (D) Biogenesis, secondary structures, and main functions of human Y RNAs. The binding sites for the Ro60 and La proteins are the cytosine bulge within the stem and the 3′oligo-uridinylated sequence, respectively. (E) Conventional functions of Y RNAs. The La protein is responsible for nuclear retention to allow control of DNA replication. Exportin 5 and 1 are required for export in the cytoplasm, where Y RNAs engage in protein interactions and affect RNA stability and processing. (F) Nonconventional mode of actions: cleavage of Y RNAs involves caspase 3 and produces functional ysRNAs, such as s-RNY1-5p, which affects apoptosis and inflammation through a TLR7-and NF-κB-dependent pathway. CCL2, CC-chemokine ligand 2; IKK, IκB kinase; IRAK1, Interleukin-1 receptor-associated kinase-1; TAB1, TAK1 binding protein 1; TAK1, TGF-β activated kinase 1; TNF-α, Tumor necrosis factor-α.
      Although molecular mechanisms of vRNAs are still under investigation, the function of vRNAs is canonically linked to the prototypical ribonucleotide complex involved in multiple pathways ranging from nucleo-cytoplasmic trafficking, scaffold of signaling proteins, to the regulation of DNA damage repair (Fig. 4B) [
      • Hahne J.C.
      • Lampis A.
      • Valeri N.
      Vault RNAs: hidden gems in RNA and protein regulation.
      ]. However, a considerable fraction of vRNAs is found outside of the “classical” vault complex and independently mediates physio-pathological processes [
      • Stadler P.F.
      • Chen J.J.
      • Hackermuller J.
      • et al.
      Evolution of vault RNAs.
      ]. For instance, vtRNA1-1 participates in regulating selective autophagy through binding to the autophagic receptor p62/SQSTM1. This interaction occurs with the ZZ-domain of the protein, prevents the p62/SQSTM1 oligomerization and its binding with LC3 in autophagosomes, thus blocking the autophagic flux (Fig. 4C) [
      • Horos R.
      • Buscher M.
      • Kleinendorst R.
      • et al.
      The small non-coding vault RNA1-1 acts as a riboregulator of autophagy.
      ]. Similar to miR-126-5p and miR-1-3p, this finding further reinforces the notion that smRNAs can function as aptamers to regulate pivotal homeostatic processes [
      • Horos R.
      • Buscher M.
      • Kleinendorst R.
      • et al.
      The small non-coding vault RNA1-1 acts as a riboregulator of autophagy.
      ,
      • Santovito D.
      • Egea V.
      • Bidzhekov K.
      • et al.
      Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.
      ,
      • Yang D.
      • Wan X.
      • Dennis A.T.
      • et al.
      MicroRNA biophysically modulates cardiac action potential by direct binding to ion channel.
      ]. Moreover, vRNAs undergo cleavage into small oligonucleotides (namely, svRNAs) through a Dicer-dependent and Drosha-independent process [
      • Persson H.
      • Kvist A.
      • Vallon-Christersson J.
      • et al.
      The non-coding RNA of the multidrug resistance-linked vault particle encodes multiple regulatory small RNAs.
      ]. A few reports describe the role of syRNAs in biological functions such as aging and neuronal damage. Yet, the mechanisms are still elusive and may go beyond AGO-dependent RNA-interference [
      • Persson H.
      • Kvist A.
      • Vallon-Christersson J.
      • et al.
      The non-coding RNA of the multidrug resistance-linked vault particle encodes multiple regulatory small RNAs.
      ,
      • Minones-Moyano E.
      • Friedlander M.R.
      • Pallares J.
      • et al.
      Upregulation of a small vault RNA (svtRNA2-1a) is an early event in Parkinson disease and induces neuronal dysfunction.
      ].
      Evidence supports the involvement of vRNAs in apoptosis and proliferation [
      • Honda S.
      • Loher P.
      • Shigematsu M.
      • et al.
      Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers.
      ]. In cancer cells, abrogation of vtRNA1-1 prevents its inhibitory role on the MAPK pathway leading to the inactivation of the transcription factor TFEB. This ultimately determines lysosomal dysfunction with consequences for proliferation and apoptosis [
      • Ferro I.
      • Gavini J.
      • Gallo S.
      • et al.
      The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma.
      ]. However, the effects on apoptosis and proliferation may partly depend on the association of vRNAs with MVP. This protein promotes growth/survival signaling in human SMCs, upon its S-glutathionylation stimulated by IL-22 or PDGF. Conversely, inhibition of MVP determines cell death through STAT3 and Akt pathways [
      • Das D.
      • Wang Y.H.
      • Hsieh C.Y.
      • et al.
      Major vault protein regulates cell growth/survival signaling through oxidative modifications.
      ] and improves remodeling of pulmonary vessels in a rat model of pulmonary arterial hypertension [
      • Wang X.
      • Ibrahim Y.F.
      • Das D.
      • et al.
      Carfilzomib reverses pulmonary arterial hypertension.
      ]. While ubiquitination and proteasomal degradation of the MVP contribute to the phenotypical determination of VSMCs in pulmonary artery hypertension [
      • Wang X.
      • Ibrahim Y.F.
      • Das D.
      • et al.
      Carfilzomib reverses pulmonary arterial hypertension.
      ], its expression is increased during aging in human fibroblasts and aged hearts, lungs, and spleens [
      • Ryu S.J.
      • An H.J.
      • Oh Y.S.
      • et al.
      On the role of major vault protein in the resistance of senescent human diploid fibroblasts to apoptosis.
      ]. Notably, deletion of Mvp in myeloid cells aggravates atherosclerosis, and mechanistic studies revealed that MVP suppresses the NF-κB pathway by averting the ubiquitination of TRAF6 (Fig. 4C) [
      • Ben J.
      • Jiang B.
      • Wang D.
      • et al.
      Major vault protein suppresses obesity and atherosclerosis through inhibiting IKK-NF-kappaB signaling mediated inflammation.
      ]. These data reveal possible indirect contributions of vRNAs in aging and atherogenesis, which are mediated by MVP. Together, these findings suggest the involvement of vRNAs in atherosclerosis and cardiovascular disease, featuring both canonical and non-canonical functions.

      5.2 Y RNAs

      Y RNAs were identified in the early 1980s as the RNA component of the La and Ro RNP autoantigens in sera of patients affected by systemic lupus erythematosus [
      • Lerner M.R.
      • Boyle J.A.
      • Hardin J.A.
      • et al.
      Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus.
      ]. However, multiple other interacting proteins were successively identified, suggesting that Y RNAs may act as scaffolds for the assembly of diverse RNPs. With their length ranging between 84 and 113 nt, Y RNAs belong to the smRNAs class and are highly conserved across species, being encoded by genes clustered in the same chromosome (in humans, chromosome 7q36) [
      • Wolin S.L.
      • Steitz J.A.
      Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome.
      ,
      • Mosig A.
      • Guofeng M.
      • Stadler B.M.
      • et al.
      Evolution of the vertebrate Y RNA cluster.
      ]. Four distinct Y RNAs have been described, namely: Y1, Y3, Y4, and Y5. They mainly differ in sequence and secondary structure as they fold into different hairpins by 3′ and 5’ pairing [
      • Teunissen S.W.
      • Kruithof M.J.
      • Farris A.D.
      • et al.
      Conserved features of Y RNAs: a comparison of experimentally derived secondary structures.
      ]. The RNA polymerase III transcribes the Y RNAs up to a poly-T region and produces a 3′ oligo-uridinylated sequence (oligo-U), which is the binding site for the protein La (Fig. 4D) [
      • Wolin S.L.
      • Cedervall T.
      The La protein.
      ]. The interaction with this protein protects the Y RNA from degradation and promotes nuclear retention. Conversely, the protein Ro60 facilitates the cytoplasmic transfer of the RNP via a Ran GTPase/Exportin-5-dependent mechanism, as described for miRNAs [
      • Simons F.H.
      • Rutjes S.A.
      • van Venrooij W.J.
      • et al.
      The interactions with Ro60 and La differentially affect nuclear export of hY1 RNA.
      ]. An alternative export pathway has been reported for Y3 and involves the binding to the zipcode binding protein (ZBP1), allowing functional interactions with Exportin-1 (Fig. 4E) [
      • Sim S.
      • Yao J.
      • Weinberg D.E.
      • et al.
      The zipcode-binding protein ZBP1 influences the subcellular location of the Ro 60-kDa autoantigen and the noncoding Y3 RNA.
      ].
      Y RNAs are reported to canonically act in concert with proteins of the Ro family, engaging in sequence- and structure-specific interactions with them. The structure of Y RNAs in vertebrates is characterized by two main stems separated by a large pyrimidine-rich single-stranded loop (Fig. 4E). A highly conserved cytosine bulge within the stem constitutes a high-affinity binding site for the protein Ro60 [
      • Green C.D.
      • Long K.S.
      • Shi H.
      • et al.
      Binding of the 60-kDa Ro autoantigen to Y RNAs: evidence for recognition in the major groove of a conserved helix.
      ]. Due to its high specificity, mutations or cleavage processes occurring at this site affect binding to the protein with abnormalities in Y RNA folding, determining functional abnormalities [
      • Green C.D.
      • Long K.S.
      • Shi H.
      • et al.
      Binding of the 60-kDa Ro autoantigen to Y RNAs: evidence for recognition in the major groove of a conserved helix.
      ]. Contrary to the structure and interfaces for protein interactions, knowledge of the functional spectrum of Y RNAs is limited. Available evidence implicates them in DNA replication, RNA stability, cell proliferation, and stress response [
      • Kowalski M.P.
      • Krude T.
      Functional roles of non-coding Y RNAs.
      ]. Yet, exact mechanisms are still elusive. Besides these traditional features, Y RNA may be cleaved into smaller RNA fragments (termed ysRNAs) by a process that does not involve DICER or the endoribonuclease RNASEL, but is apparently dependent on caspase 3 (Fig. 4F) [
      • Billmeier M.
      • Green D.
      • Hall A.E.
      • et al.
      Mechanistic insights into non-coding Y RNA processing.
      ].
      Although Y RNAs are detectable in all human cells, their expression is cell- and tissue-specific, with higher abundance measured in brain and heart compared to liver [
      • Pruijn G.J.
      • Wingens P.A.
      • Peters S.L.
      • et al.
      Ro RNP associated Y RNAs are highly conserved among mammals.
      ]. As expected for molecules involved in cellular stress response, aberrant expression of Y RNAs has been associated with diseases, such as cancer and cardiovascular diseases [
      • Repetto E.
      • Lichtenstein L.
      • Hizir Z.
      • et al.
      RNY-derived small RNAs as a signature of coronary artery disease.
      ]. In particular, s-RNY1-5p (a ysRNA processed from Y1) is upregulated in the aortic arch of atheroprone Apoe−/− and Ldlr−/− mice and further increased by a high-fat diet. Likewise, s-RNY1-5p is stimulated by pro-atherogenic stimuli in macrophages and VSMCs in vitro [
      • Repetto E.
      • Lichtenstein L.
      • Hizir Z.
      • et al.
      RNY-derived small RNAs as a signature of coronary artery disease.
      ]. Mechanistic studies in atherosclerosis are still to be conducted, however these smRNAs promote apoptosis and activate the NF-κB inflammatory pathway in macrophages through a Toll-like receptor (TLR)7-dependent mechanism [
      • Hizir Z.
      • Bottini S.
      • Grandjean V.
      • et al.
      RNY (YRNA)-derived small RNAs regulate cell death and inflammation in monocytes/macrophages.
      ]. Finally, ysRNAs are released in the serum conveyed by proteins, and circulating levels of s-RNY1-5p are higher in patients with coronary artery disease [
      • Repetto E.
      • Lichtenstein L.
      • Hizir Z.
      • et al.
      RNY-derived small RNAs as a signature of coronary artery disease.
      ], possibly serving as a disease biomarker.

      6. Long non-coding RNAs and circular RNAs

      Contrary to smRNAs, lncRNAs are a heterogeneous class of ncRNAs that widely differ in function, expression, subcellular localization, and size (from 200 nt to 100 kb) [
      • Statello L.
      • Guo C.J.
      • Chen L.L.
      • et al.
      Gene regulation by long non-coding RNAs and its biological functions.
      ]. Despite being similar to mRNAs at the molecular level, lncRNAs possess ORFs shorter than 300 nt, thus lacking the coding potential of mRNAs. Yet, they might be translated into micropeptides [
      • Xing J.
      • Liu H.
      • Jiang W.
      • et al.
      LncRNA-encoded peptide: functions and predicting methods.
      ]. The vast majority of lncRNAs are transcribed by RNA polymerase II and III, processed by the spliceosome, and have 5ʹ-caps and 3′ polyadenylated tails [
      • Statello L.
      • Guo C.J.
      • Chen L.L.
      • et al.
      Gene regulation by long non-coding RNAs and its biological functions.
      ]. Mature transcripts are low-copy number molecules with specific subcellular localization [
      • Statello L.
      • Guo C.J.
      • Chen L.L.
      • et al.
      Gene regulation by long non-coding RNAs and its biological functions.
      ,
      • Cabili M.N.
      • Dunagin M.C.
      • McClanahan P.D.
      • et al.
      Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution.
      ]. The expression of lncRNAs is regulated in a tissue-specific manner during embryogenesis and cell cycle, underscoring their regulatory relevance during development. Depending on their directionality with respect to coding genes, lncRNAs are defined as “sense” (S) or “antisense” (AS), although they also originate from exonic, intronic, intergenic (linc), enhancer (e), promoter, or 3′UTR regions (Fig. 5A) [
      • Statello L.
      • Guo C.J.
      • Chen L.L.
      • et al.
      Gene regulation by long non-coding RNAs and its biological functions.
      ].
      Fig. 5
      Fig. 5Long non-coding RNAs (lncRNAs).
      (A) Classification of lncRNAs by their biogenesis. (B) Mechanisms of gene regulation in cis- and trans. By binding to transcription factors (TFs), lncRNAs may act as signals to start transcription or as decoys to prevent gene expression. LncRNAs tether to epigenetic enzymes to scaffold or drive them toward genomic loci, thus allocating epigenetic marks for transcriptional activation (e.g., H3K27ac and H3K4me3) or inhibition (e.g., H3K27me3). LncRNAs can mediate the interactions between promoters and enhancers, thus fostering gene transcription. In the cytoplasm, lncRNAs can bind to ribosomes to activate or inhibit protein translation. Finally, lncRNAs may sponge miRNAs and hinder their functionality. (C) The interaction of lncRNAs with proteins serves as a scaffold for the assembly of RNPs or may affect the functionality of intracellular signaling pathways (e.g., Notch1 pathway). (D) Backsplicing is a non-canonical splicing event linking downstream 5′ donor to upstream 3′ acceptor sites to generate circRNAs. Mainly located in the cytoplasm, circRNAs act as sponges for proteins (e.g., PES1, SDOS) or miRNAs (e.g., miR-145-5p) and influence their functions.
      The conservation of lncRNAs across species is limited, nevertheless many conserved structural properties suggest an evolutionary functional relevance for some of them [
      • Palazzo A.F.
      • Lee E.S.
      Non-coding RNA: what is functional and what is junk?.
      ]. On the contrary, the promoters of lncRNAs are evolutionarily conserved and histone modifications that determine activity, such as H3K4me3 or H3K27ac, are the same as for promoters of protein-coding genes. Many promoters are bidirectional and simultaneously produce sense and antisense RNA transcripts with similar expression levels [
      • Seila A.C.
      • Calabrese J.M.
      • Levine S.S.
      • et al.
      Divergent transcription from active promoters.
      ]. However, while sense transcripts are enriched in splice sites, antisense transcripts are characterized by polyadenylation signals that determine early RNA termination [
      • Statello L.
      • Guo C.J.
      • Chen L.L.
      • et al.
      Gene regulation by long non-coding RNAs and its biological functions.
      ]. Most lncRNAs are divergent transcripts that regulate their own promoters and the ones of the corresponding protein-coding genes in cis [
      • Engreitz J.M.
      • Haines J.E.
      • Perez E.M.
      • et al.
      Local regulation of gene expression by lncRNA promoters, transcription and splicing.
      ]. This includes lncRNAs of imprinted loci and X chromosome inactivation, such as Xist (X-inactive specific transcript), which is transcribed from one of the X chromosomes in placental mammals and is the main player in sex-chromosome dosage compensation [
      • Autuoro J.M.
      • Pirnie S.P.
      • Carmichael G.G.
      Long noncoding RNAs in imprinting and X chromosome inactivation.
      ]. On the other hand, some lncRNAs function far away from their transcriptional site, either in the cytoplasm or in other cellular compartments, and are therefore referred to as trans-acting lncRNAs (Fig. 5B) [
      • Yan P.
      • Luo S.
      • Lu J.Y.
      • et al.
      Cis- and trans-acting lncRNAs in pluripotency and reprogramming.
      ]. The regulation of gene expression in cis and trans are considered the major mode of function of lncRNAs. However, growing evidence depicts additional peculiar features (e.g., protein binding, circularization) which extend the functional repertoire of lncRNAs and allow wide regulation of biological processes.
      The contribution of lncRNAs to vascular biology is an active research topic, and evidence has accumulated on the importance of this class of ncRNAs in atherogenesis. In particular, the lncRNA ANRIL (antisense ncRNA in the INK4 locus) guides PRC1 and PRC2 complexes, thus regulating the promoter activity of target genes [
      • Holdt L.M.
      • Hoffmann S.
      • Sass K.
      • et al.
      Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks.
      ]. The expression of ANRIL is modulated during atherogenesis and this lncRNA acts both in cis, by enriching the repressive histone mark H3K27me3 in the promoter of the CDKN2B gene [
      • Kotake Y.
      • Nakagawa T.
      • Kitagawa K.
      • et al.
      Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15(INK4B) tumor suppressor gene.
      ], and in trans by mirroring the Alu motifs in its sequence to genomic regions of genes involved in cell adhesion, proliferation and apoptosis, which are pathways crucially involved in atherosclerosis [
      • Holdt L.M.
      • Hoffmann S.
      • Sass K.
      • et al.
      Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks.
      ]. Notably, the gene encoding for ANRIL maps in chromosome 9p21, one of the most replicated loci for its association with coronary artery disease in genome-wide association studies [
      • Holdt L.M.
      • Hoffmann S.
      • Sass K.
      • et al.
      Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks.
      ]. Another trans-acting lncRNA is HOTAIR (HOX transcript antisense intergenic RNA) which guides PRC2 to the HOXD locus far from the region of its transcription [
      • Gupta R.A.
      • Shah N.
      • Wang K.C.
      • et al.
      Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.
      ]. HOTAIR is reduced in human atherosclerotic plaques compared to healthy vessels, and mechanistic experiments in vitro revealed its ability to improve endothelial function in response to atherogenic stimuli, through regulation of the PI3K/AKT-IRF1 pathway [
      • Peng Y.
      • Meng K.
      • Jiang L.
      • et al.
      Thymic stromal lymphopoietin-induced HOTAIR activation promotes endothelial cell proliferation and migration in atherosclerosis.
      ]. Furthermore, lncRNAs are crucial for the phenotype of VSMCs. The lncRNA MIAT (Myocardial infarction-associated transcript) mediates the phenotypic switch of VSMCs by regulating KLF4 transcription and promotes their transdifferentiation toward a macrophage-like state via the EGR1-ELK1-ERK pathway [
      • Fasolo F.
      • Jin H.
      • Winski G.
      • et al.
      Long noncoding RNA MIAT controls advanced atherosclerotic lesion formation and plaque destabilization.
      ]. On the other hand, the VSMC-enriched CARMN (cardiac mesoderm enhancer-associated ncRNA) gained interest in recent years due to its location upstream of the MIR143/145 gene, encoding for miR-143 and miR-145 which are major determinants of the VSMC phenotype. Recent data indicate that CARMN also binds the VSMC-specific transcriptional cofactor myocardin and stabilizes the contractile phenotype [
      • Dong K.
      • Shen J.
      • He X.
      • et al.
      CARMN is an evolutionarily conserved smooth muscle cell-specific LncRNA that maintains contractile phenotype by binding myocardin.
      ].
      Besides transcriptional regulation and epigenetics, lncRNAs may also sequester miRNAs and influence the expression of their putative target transcripts. This is the case of MALAT1 (also known as NEAT2) which sponge multiple miRNAs (e.g., miR-22-3p, miR-26b-5p, miR-145, and miR-155) with regulatory roles on biological processes important for atherosclerosis, such as proliferation, apoptosis, autophagy, and pyroptosis [
      • Wang Q.
      • Lu G.
      • Chen Z.
      MALAT1 promoted cell proliferation and migration via MALAT1/miR-155/MEF2A pathway in hypoxia of cardiac stem cells.
      ,
      • Li R.
      • Yan G.
      • Li Q.
      • et al.
      MicroRNA-145 protects cardiomyocytes against hydrogen peroxide (H(2)O(2))-induced apoptosis through targeting the mitochondria apoptotic pathway.
      ,
      • Song T.F.
      • Huang L.W.
      • Yuan Y.
      • et al.
      LncRNA MALAT1 regulates smooth muscle cell phenotype switch via activation of autophagy.
      ,
      • Tang Y.
      • Jin X.
      • Xiang Y.
      • et al.
      The lncRNA MALAT1 protects the endothelium against ox-LDL-induced dysfunction via upregulating the expression of the miR-22-3p target genes CXCR2 and AKT.
      ]. The protective role of MALAT1 against atherosclerosis in myeloid and endothelial cells emerged from recent studies [
      • Leucci E.
      • Patella F.
      • Waage J.
      • et al.
      microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus.
      ,
      • Cremer S.
      • Michalik K.M.
      • Fischer A.
      • et al.
      Hematopoietic deficiency of the long noncoding RNA MALAT1 promotes atherosclerosis and plaque inflammation.
      ], and a genetic variant (rs3200401) within the MALAT1 locus is associated with incident cardio- and cerebrovascular events, thus translating these findings to humans [
      • Zhang T.
      • Luo J.Y.
      • Liu F.
      • et al.
      Long noncoding RNA MALAT1 polymorphism predicts MACCEs in patients with myocardial infarction.
      ]. Finally, lncRNAs also interact with proteins and influence intracellular signaling pathways (Fig. 5C). In the example, lncWDR59 establishes a repressive interaction with the protein NUMB, an inhibitor of Notch1, enhancing the activity of the Notch1 pathway, preserving endothelial proliferative reserve, and preventing the progression of atherosclerosis [
      • Natarelli L.
      • Geissler C.
      • Csaba G.
      • et al.
      miR-103 promotes endothelial maladaptation by targeting lncWDR59.
      ].
      Very intriguingly, RNAs may be processed into covalently closed loop structures termed circular RNAs (circRNAs) (Fig. 5D). They are one of the most recently discovered classes of ncRNAs which is abundantly expressed in many eukaryotic tissues in a time-dependent manner [
      • Veno M.T.
      • Hansen T.B.
      • Veno S.T.
      • et al.
      Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development.
      ]. CircRNAs originate from canonical splicing sites through a non-canonical splicing event, termed backsplicing, that covalently links a downstream 5'splice-donor site to an upstream 3'splice-acceptor site [
      • Kristensen L.S.
      • Andersen M.S.
      • Stagsted L.V.W.
      • et al.
      The biogenesis, biology and characterization of circular RNAs.
      ]. Being circular structures, they lack extremities and, consequently, 5ʹcaps and 3′polyadenylated tails. However, they prevalently localize in the cytoplasm where they act as sponges for RNA-binding proteins or miRNAs. The involvement of circRNAs in atherosclerosis emerged in recent years. A circularized form of ANRIL (circANRIL) originates from backsplicing of exons 7, 5 and 6 of ANRIL and it is expressed in VSMCs and CD68+ cells of human atherosclerotic plaques. Mechanistically, circANRIL binds to the C-terminal of the protein PES1, thus preventing exonuclease-mediated rRNA maturation and ribosome biogenesis. The resulting defective ribosome assembly affects apoptosis and proliferation of VSMCs through a p53-dependent pathway [
      • Holdt L.M.
      • Stahringer A.
      • Sass K.
      • et al.
      Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans.
      ]. Besides apoptosis and proliferation, circRNAs also regulate phenotypic switch of VSMCs. In particular, circLRP6 is endowed with multiple binding sites for miR-145-5p and acts as an endogenous sponge hindering the ability of this miRNA to maintain a contractile phenotype, thereby favoring atherogenesis [
      • Hall I.F.
      • Climent M.
      • Quintavalle M.
      • et al.
      Circ_Lrp6, a circular RNA enriched in vascular smooth muscle cells, acts as a sponge regulating miRNA-145 function.
      ]. Finally, circRNAs are also relevant to EC biology and contribute to their response to shear stress. Among them, circZfp292 is a locus-conserved circRNA and interacts with the protein syndesmos (SDOS), an RNA-binding involved in cytoskeleton organization and focal-adhesion signaling. Deletion of circZfp292 in vivo inhibits the physiological alignment of aortic ECs to the flow, thus suggesting a possible protective role of this circRNA in areas exposed to laminar shear stress [
      • Heumuller A.W.
      • Jones A.N.
      • Mourao A.
      • et al.
      Locus-conserved circular RNA cZNF292 controls endothelial cell flow responses.
      ].

      7. Non-coding functions of coding RNAs

      Recent research shows how the boundary among RNA classes, particularly concerning their intrinsic ability to encode proteins, is not always sharp. Whilst lncRNAs are reported to encode small peptides through atypical open-reading frames [
      • Xing J.
      • Liu H.
      • Jiang W.
      • et al.
      LncRNA-encoded peptide: functions and predicting methods.
      ], mRNAs displayed a variety of functions unrelated to their coding potential. Wide portions of mRNA do not contribute to the protein sequence, yet they exert regulatory roles. For instance, 3′UTRs host multiple MREs for miRNA-dependent post-transcriptional regulation [
      • Bartel D.P.
      Metazoan MicroRNAs.
      ], 5′UTRs regulate translation by folding into secondary/tertiary structures to affect ribosome recruitment [
      • Leppek K.
      • Das R.
      • Barna M.
      Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.
      ], while introns may act as architectural RNAs (arcRNAs). This latter feature is epitomized by introns 3 and 5 of the SRSF7 transcript. They mediate the assembly of nuclear bodies to sequester the SRSF7 protein and fully-spliced mRNA, realizing a feedback mechanism to control cytoplasmic SRSF7 protein [
      • Konigs V.
      • de Oliveira Freitas Machado C.
      • Arnold B.
      • et al.
      SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly.
      ]. Auto-regulatory roles are also reported for TP53 mRNA, which interacts with the protein MDM2 and prevents proteasomal degradation of the p53 protein, or acts as an intracellular sensor to regulate TP53 transcription and splicing [
      • Candeias M.M.
      The can and can't dos of p53 RNA.
      ]. Finally, mRNAs play architectural functions in the cytoplasm. Indeed, multiple mRNAs anchor to focal adhesions of actin cytoskeleton to extracellular matrix. These transcripts remain translationally inactive and shape a network of proteins to regulate EC adaptation to the extracellular matrix [
      • Boraas L.
      • Hu M.
      • Thornton L.
      • et al.
      Non-coding function for mRNAs in focal adhesion architecture and mechanotransduction.
      ]. The non-coding functions of mRNAs are subject to intense investigation, and other regulatory pathways are likely to emerge. This would offer new opportunities to fine-tune protein expression to control complex biological processes such as matrix-cell interactions.

      8. Outlook and translational perspective

      The last two decades showed how the development of cutting-edge technologies (e.g., next-generation sequencing) has revolutionized the research field allowing annotation and of many ncRNAs, characterization of their biogenesis, specifying their localization, and defining their functions. Studies mainly focused on their role in transcriptional/posttranscriptional regulation, while the emergence of the novel modes of action described in this Review is more recent. At this stage, we appreciate some non-canonical functions (e.g., biogenesis of smaller functionally active fragments, protein-interaction ability) executed by diverse ncRNA classes, owning structural features which only resemble their originating DNA sequence as they undergo deep processing of RNA molecules. Yet, the occurrence of several non-canonical functions (e.g., aptamer function of miR-1-3p and miR-126-5p, circularization of lncRNAs, architectural role of retained introns) appears specific for some ncRNAs and is likely licensed by sequence motifs or secondary structures. To date, no clear similarity patterns have been identified, and their study is an active research area and will be facilitated by technical and technological advances. Among them, high-resolution spatial transcriptomics will enable ncRNA mapping at the subcellular level, allowing the identification of atypical localization possibly featuring distinctive functional roles. Moreover, crosslinking and immunoprecipitation approaches (CLIP) have evolved to detect protein-RNA interfaces at single-nucleotide resolution (e.g., eCLIP, iCLIP) and will contribute to extending our knowledge on aptamer functions of ncRNAs. Finally, the enhancement of structural (e.g., cryo-electron microscopy) and chemical probing approaches (e.g., SHAPE), as well as progresses of artificial intelligence in learning and predicting protein and RNA folding (e.g., AlphaFold, ARES) will contribute to a comprehensive understanding of structural bases of functional ncRNA-protein interactions.
      Given their role as mediators in atherogenesis and cardiovascular diseases, ncRNAs are not only potential non-invasive diagnostic/prognostic biomarkers but also an excellent therapeutic opportunity. The recent clinical approval of inclisiran as a siRNA to repress PCSK9 expression represents a milestone in cardiovascular therapy [
      • Ray K.K.
      • Wright R.S.
      • Kallend D.
      • et al.
      Two phase 3 trials of inclisiran in patients with elevated LDL cholesterol.
      ] and opens up new horizons to the future application of ncRNAs (not limited to siRNAs) as therapeutic agents. This Review has examined numerous ncRNAs aberrantly regulated during atherogenesis which may represent targets for future RNA-based therapeutics. Remarkably, the occurrence of non-conventional mechanisms of action of ncRNAs may represent an opportunity for tailored targeting of specific molecular processing (e.g., aptamer-like interactions) without affecting the expression of genes or ncRNA, which may also result in conflicting effects. An extensive clarification of these mechanisms as well as the development of approaches for selective manipulation and delivery are the next challenges in this research field and may lead to the development of future optimal therapeutic strategies for atherosclerosis and beyond.

      Declaration of competing interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgments

      The work of the authors is supported by the Deutsche Forschungsgemeinschaft (DFG, Project IDs: 403584255 - TRR 267-A2, SFB1123-A1/A10 to C.W., and SFB1123–B5 to D.S.), by the German Society for Cardiovascular Research (DZHK, project 81X2600269 to D.S.), the Bundesministerium für Bildung und Forschung (BMBF) and Free State of Bavaria (LMU Excellence strategy to C.W. and D.S.), and by the European Research Council (ERC AdG 692511 to C.W.). C.W. is a Van de Laar professor of atherosclerosis.

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