Noncoding RNAs in cardiovascular biology and disease.

Abstract

HomeCirculation ResearchVol. 113, No. 12Noncoding RNAs in Cardiovascular Biology and Disease Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBNoncoding RNAs in Cardiovascular Biology and Disease Priyatansh Gurha and Ali J. Marian Priyatansh GurhaPriyatansh Gurha From the Center for Cardiovascular Genetics, Institute of Molecular Medicine and Department of Medicine, University of Texas Health Sciences Center at Houston, and Texas Heart Institute, Houston, TX. Search for more papers by this author and Ali J. MarianAli J. Marian From the Center for Cardiovascular Genetics, Institute of Molecular Medicine and Department of Medicine, University of Texas Health Sciences Center at Houston, and Texas Heart Institute, Houston, TX. Search for more papers by this author Originally published6 Dec 2013https://doi.org/10.1161/CIRCRESAHA.113.302988Circulation Research. 2013;113:e115–e120The genome continues to fascinate the enthusiasts, and the captivation seems unabating because of the continuous stream of new discoveries. What was once considered a junk DNA has now emerged to contain a large number of important regulatory elements.1 As an example of such discoveries is the recent finding that the genome contains ≈6200 enhancer elements that are operational in the human fetal and adult hearts.2 Genes occupy only ≈1.5% and coding exons only ≈1% of the genome, which make up only ≈60 million nucleotides of the 6.4 billion nucleotides (3.2 billion base pairs) in the genome.3 Yet, ≈5% of the human genome has undergone purifying selection and hence are likely functional.4 It is intriguing that about two third of the evolutionary constrained genomic elements are located in introns and the intergenic regions, suggestive of their regulatory roles in the genome.4 These conserved regions are enriched in loci that have been found to be associated with clinical phenotypes in the genome-wide association studies.4 The initial findings of the ENCODE (Encyclopedia of DNA Elements) project, although preliminary, illustrate the presence of numerous regulatory elements in the genome, including the enhancers.1The discoveries largely made possible by the recent advances in the high-throughput RNA sequencing point to enormous RNA splicing diversity and the plethora of alternative splicing variants. Approximately 95% of the multiexon genes undergo alternative splicing, resulting in ≈100 000 abundant splice variants in various tissues.5 Moreover, it seems that almost all genomic regions in 1 form or shape are transcribed, which seems perplexing as only ≈1% of the genome codes for proteins, as has been understood to date. Only recently we have started to appreciate the diverse biological functions of these nonprotein coding transcripts, which are referred to as noncoding RNAs (ncRNAs). And yet, it is mesmerizing to learn that ncRNAs might indeed contain small open reading frames and encode functional peptides.6,7 The long ncRNA (lncRNA) pncr003:2L in Drosophila encodes two 28 and 29 amino acid peptides sarcolamban A and B because of their structural and functional similarities to sarcolipin and phospholamban, regulators of Ca+2 uptake by SERCA2 and cardiac function.6 Thus, in a sense, the term ncRNAs might be a misnomer for some ncRNAs, because they might code for small peptides. If this discovery turns out to be a common feature of the lncRNAs, the discovery has the potential to change the landscape of genomic biology and medicine dramatically.The rRNA, tRNA, snRNA, and snoRNA were among the first ncRNAs to be identified and characterized to have a role in mRNA translation and RNA processing events such as nucleotide modification and splicing.8 The discovery of Lin-4, the first microRNA (miRNA), in Caenorhabditis elegans by Ambros and colleagues9 in 1993 ushered in a new era and was soon followed by identification of new classes of ncRNA, primarily based on whole transcriptome sequencing (Table 1). These discoveries expanded the function of ncRNAs as fine regulators of various biological processes. It is now evident that ncRNAs mediate both post-transcriptional and transcriptional gene regulation, predominantly through RNA-guided (dependent) mechanisms.10 Moreover, recent data also suggest that miRNAs have autoregulatory functions.11,12Table 1. Classification and Function of Noncoding RNAsRNASize, NucleotidesFeatures and FunctionSmall noncoding RNAmiRNAs21–24RNA polymerase (Pol) II transcribed. Primary transcript is processed to mature miRNA mostly by DROASHA and DICER. They regulate post-transcriptional gene expressiontRNA-derived small RNAs17–22RNA Pol III transcribed. Generated by 5′ and 3′-end processing of tRNAs. Function similar to miRNAssnoRNA-derived RNAs20–25Pol III transcribed. Generated from snoRNA processing independent of Drosha. Function similar to miRNAsmoRNA≈20RNAs derived from the ends of pre-miRNAs, possibly by RNase III–like processing. Function not clearY-RNA–derived RNA24–25RNA fragments derived from Y-RNA. Processed independent to miRNA pathway. Function not defined; may be involved in autoimmunityEndogenous siRNA17–21Intergenic or transcribed from repeat cluster. Function to silence genespiRNA24–30Pol II transcribed. Mature piRNA binds to PIWI or MIWI protein; gets 2′-O-Me (methylation) at 3′-end. Mediate transposon silencing and gene regulationspliRNA17–18Pol II transcribed. 3′-end of these RNAs map to the splice donor sitetiRNA≈18Generated by Pol II. Produced as both sense and antisense RNAs from sequences +250 to −50 to start site. Epigenetic regulation of gene expressionTranscription start site–associated RNA20–90Pol II transcribed. Contains 5′ cap generated at or near the transcription start siteTermini-associated small RNA15–30Pol II transcribed. Contains 5′ cap and nongenome-encoded 5′ polyU sequences. Generated from 3′-untranslated region of coding genestRNA70–90Pol III transcribed. Function in mRNA translation, signal transduction, apoptosis, etcY-RNA80–110Pol III transcribed; 4 types in human required for DNA replicationLong noncoding RNAsnRNA90–200Pol II and III transcribed. RNA component of spliceosome. Required for pre-mRNA splicingsnoRNA70–200Two classes, namely C/D box RNA and H/ACA box RNA. Function in rRNA processing and rRNA, tRNA, and snRNA nucleotide modificationH1 RNA≈360Pol III transcribed. Component of RNase P. Required for tRNA 5′-end processing and Pol III transcriptionTelomerase RNA component≈450Pol III transcribed. RNA component of telomerase. Act as template for telomeric DNA synthesisPromoter-associated long noncoding RNA<200Synthesized by Pol II. Contains 5′ cap and 3′ poly(A) tail. Function in transcriptional activation and silencingEnhancer RNA (eRNA)Gene regulation in cis or trans through chromatin modificationUnidirectionaal eRNA0.5–2 kbPol II transcribed. Contains 5′ cap but no 3′ poly(A) tailBidirectional eRNA≈3–4 kbPol II transcribed. Contains 5′ cap and 3′ poly(A) tailLong intervening noncoding RNAs<200Mainly Pol II transcribed. Mostly contains 5′ cap and 3′ poly(A) tail similar to mRNAs. Function in gene activation and silencing by acting in cis or transClustered regularly interspaced short palindromic repeat RNA61Found in bacteria and archaea only. Act as surveillance mechanism against viruses. Function in association with Cas1 protein and target foreign nucleic acids for silencingtmRNA≈350Function both as tRNA and mRNA. tmRNA rescue stalled ribosomes by mRNA swapping and tagging protein for degradationThe current classification of ncRNAs is based primarily on transcript size as small ncRNAs (<200 nucleotides) and lncRNAs (>200 nucleotides). Small ncRNAs consist of several diverse arrays of RNAs ranging in size from 17 to 200 nucleotides that follow distinct path for their biogenesis and function (Table 1). A notable member in the class of ncRNAs is miRNAs, which are ≈22 nucleotides long and orchestrate post-transcriptional gene regulation (as RNA protein complex) through base pairing to their target RNAs and either degradation of the mRNA or inhibition of its translation. miRNAs are generally produced as RNA polymerase II–transcribed primary transcript, namely pri-miRNA. The biogenesis of pri-miRNA transcript occurs either through the canonical pathway involving DROSHA and DICER (RNase type III enzyme) or through various noncanonical pathways that are DROSHA- and even DICER-independent.13–15 Likewise, recent data show that miRNAs could be produced from snoRNA, tRNA, or Y-RNA, as intermediate products.16–18miRNAs, first discovered in worms (Lin-4) ≈20 years ago, are now known to have a role in many biological processes in mammals.9,19 The importance of miRNAs in cardiac development, biology, and physiology was initially demonstrated by cardiac deletion of Dicer1 at different stages of development.20,21 Conditional deletion of Dicer during development (E8.5) could lead to embryonic lethality, whereas deletion at early age (3 weeks) could lead to cardiac arrhythmia. Deletion at older age (8 weeks) could lead to heart failure with ventricular enlargement, fibrosis, and cardiac myocyte disarray.22,23 Several expression-based studies in human diseased hearts and animal models, along with gain- and loss-of-function studies in mice, have shown pathogenic and protective functions of miRNAs in vivo.24–34 These discoveries have established the importance of miRNAs in regulating cardiovascular development, hypertrophy, contractility, fibrosis, apoptosis, valve formation, and gene expression in the heart.28,30,35,38–49 Recently, miRNA-24 has been shown to be a finetuner of the excitation/contraction coupling through regulation of junctophilin-2 at the dyad in heart.44,50 In addition to myocardial biology, miRNAs have a prominent role in vascular biology and are known to modulate gene expression and cell fate in smooth muscle and endothelial cells.36,37,39,43,51–55 For example, miRNA-143/145 cluster is involved in the regulation of contractile smooth muscle cell phenotype through targeting KLF4, ELK1, and CAMKII δ.56 Similarly, miRNA-17–92 cluster and miRNA-126 regulate angiogenesis and endothelial cell function.57 In addition, age-related downregulation of extracellular matrix proteins has been associated with increased expression of miRNA-29 in aneurysmal aortic dilatation.58,59 miRNAs also regulate mitochondrial function, metabolism, and cholesterol biosynthesis. miRNA-10b regulates reverse cholesterol transport, partly through interaction with gut microbiata.42,60,61 A remarkable discovery with notable implications is the discovery of cardiac miRNA 208-a regulating systemic energy homeostasis and body weight through targeting MED13 subunit of the mediator complex, which controls transcription by nuclear hormone receptors.62,63 The ability of miRNAs to determine pluripotency, lineage commitment, reprogramming, proliferation, and differentiation has set forth the potential use of miRNAs in cardiac repair and regeneration.64–68 New areas of research in cardiac regeneration have now focused on the use of miRNAs for direct and indirect in vivo reprogramming. A combination of miRNAs-1, -133, -208, and -499 has been used in the proof-of-concept for in vivo and in vitro direct reprogramming of cardiac fibroblasts to cardiac myocytes.45,69 Recently, miRNA-15 and miRNA-17–92 families have been identified as regulators of cardiac myocyte mitotic arrest and proliferation.65,70 Similarly, miRNA-590-3p and miRNA-199-3p were identified through high-throughput screening and shown to regulate neonatal cardiac myocyte proliferation, as indicated by DNA synthesis and increased cytokinesis.71,72 The fields of miRNAs have now moved from expression-based studies to define the role of subsets of miRNAs and their targetomes.73 Not surprisingly, miRNAs are now considered as therapeutic reagents to promote cardiac myocyte re-entry into the cell cycle and improve cardiac function in the diseased heart.73–77 Finally, in addition to the resident miRNAs, circulating miRNAs are also emerging as potential biomarkers and extracellular communicators, which target recipient cells and potentially regulate translation in host cells.78miRNAs represent just 1 class of small ncRNAs. The characterization of additional small ncRNAs, namely piRNAs, spliRNAs, tiRNAs, along with their associated proteins in the cardiovascular system, is expected to open new avenues for gene regulation/modulation. Such discoveries are expected to offer new dimensions to the exciting world of gene regulation and small RNA biology in the cardiovascular system.The advent of high-throughput sequencing technologies coupled with mass spectrometry and bioinformatics techniques has led to the discovery of lncRNAs and hence expanded the field of ncRNAs dramatically. lncRNAs range in size from 200 bp to a hundred kilobases. Computational analysis identified ≈7000 lncRNAs in the human genome.79 The NONCODE database has catalogued 73 327 lncRNAs from various organisms, including 33 788 from humans (http://159.226.118.44/NONCODERv3/index.htm). lncRNAs have been classified into multiple groups based on their genomic location, such as intergenic/intervening RNAs (lincRNA) and intronic or exonic lncRNAs. lncRNAs are transcribed by RNA polymerase II, and most of them undergo alternative splicing, 5′-capping, and polyadenylation. They could also serve as a template for transcription of small ncRNAs.80 Mature lncRNAs are thought to have low protein coding potential, because they lack known protein coding domains or open reading frames, display random codon usage, and have no significant bias toward silent nucleotide substitutions. Furthermore, lncRNAs are under less selective pressure and, therefore, show less sequence conservation.81,82 However, as discussed earlier, at least a subgroup of lncRNAs also might code for small peptides. The lncRNA pncr003:2 L encodes sarcolamban A and B, which are small peptides involved in regulating Ca+2 uptake and cardiac contractility.6,7,83 Discoveries on the coding potential of lncRNAs, which suggest a dual functional role for lncRNAs, if ubiquitous rather than a rare event of nature, have the potential to shift the paradigms in molecular biology and medicine.Mechanistically, lncRNAs might function either in cis or trans to modulate the expression of their target genes by using a wide range of molecular mechanisms, such as serving as a scaffold for recruitment of chromatin modifiers or transcription factors, or as decoys for protein sequestration and miRNA sponges to activate or silence genes.82,84–87 Furthermore, lncRNAs have also been reported to influence mRNA splicing, translation, and turnover.88–90 In the heart, a few lncRNAs have been implicated in regulating cardiac lineage commitment and cardiac development, which underscore the possibility that lncRNAs represent new modes of developmental regulation (Table 2). The lncRNA Braveheart regulates expression of cardiovascular genes through targeting the mesoderm posterior 1 transcription factor.91,92 It also binds to the PRC2 complex and influences epigenetic regulation of gene expression.91 The lncRNA Fendrr (FOXF1 adjacent noncoding developmental regulatory RNA) also binds to PRC2 and TRXG/MLL complexes and regulates transcription factors involved in cardiac mesoderm differentiation.93Table 2. Long Noncoding RNA in Heart DiseaseRNAFeature/Function7SKEndogenous inhibitor of cyclin T/Cdk. Controls cardiac hypertrophy100,101Braveheart (Bvht)Required for cardiogenesis by epigenetic regulation of cardiac gene expression through PRC2 complex91Foxf1 adjacent noncoding developmental regulatory RNA (Fendrr)Regulator of heart development. Regulates Foxf1 expression in cis and other targets through PRC2 and other epigenetic modifying protein93CDKN2B-AS (ANRIL)Synthesized from 9p21 locus and identified as genetic susceptibility locus for coronary disease through genome-wide association studies95,96Myocardial infarction–associated transcript (MIAT)Identified through genome-wide association studies as risk factor for myocardial infarction102Myh7-AsAntisense RNA to Myh7 mRNA, associated with reciprocal expression to Myh7. Mechanism of action unknown94Another class of lncRNAs that is complementary to other endogenous RNAs is called natural antisense transcripts (NATs). They can be transcribed in cis from opposing DNA strands at the same genomic locus (cis-NATs) or in trans from separate loci (trans-NATs). These RNAs regulate corresponding sense mRNAs by transcriptional silencing, imprinting, splicing, or editing. Two of the better-characterized NATs with role in cardiac pathophysiology are the myosin heavy chain antisense RNA transcript (bII-NAT) and the antisense ncRNA CDKN2B-AS (ANRIL).94–96 Whereas the former is coregulated with myosin heavy chain genes during neonatal development,97 the latter is an inflammation-responsive lncRNA that targets cell cycle regulator CDKN2B and hence regulates smooth muscle cell proliferation and senescence.98In view of these recent developments, one might speculate that almost all processes of cardiac pathobiology are governed by ncRNAs. Whereas miRNAs have emerged as the major tweakers and nudgers of the genome management,99 it remains to be seen whether lncRNAs, similar to their smaller-sized counterparts, are more of finetuners or major regulator of the genome. Genomic discoveries are expected to continue to fascinate enthusiasts in the years to come.Recent Developments in Cardiovascular Research: The goal of “Recent Developments” is to provide a concise but comprehensive overview of new advances in cardiovascular research, which we hope will keep our readers abreast of recent scientific discoveries and facilitate discussion, interpretation, and integration of the findings. This will enable readers who are not experts in a particular field to grasp the significance and impact of work performed in other fields. It is our hope and expectation that these “Recent Developments” articles will help readers to gain a broader awareness and a deeper understanding of the status of research across the vast landscape of cardiovascular research.—The EditorsDisclosuresNone.FootnotesCorrespondence to Ali J. Marian, MD, 6770 Bertner St, Suite C900A, Houston, TX 77030. E-mail [email protected]