Metazoan tRNA introns generate stable circular RNAs in vivo.
Ontology highlight
ABSTRACT:
SUBMITTER: Lu Z
PROVIDER: S-EPMC4536317 | biostudies-literature | 2015 Sep
REPOSITORIES: biostudies-literature
Publications
Metazoan tRNA introns generate stable circular RNAs in vivo.
Lu Zhipeng Z Filonov Grigory S GS Noto John J JJ Schmidt Casey A CA Hatkevich Talia L TL Wen Ying Y Jaffrey Samie R SR Matera A Gregory AG
RNA (New York, N.Y.) 20150720 9
We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system f ...[more]
Similar Datasets
Project description:Human survival motor neuron 1 (SMN1) codes for SMN, an essential housekeeping protein involved in most aspects of RNA metabolism. Deletions or mutations of SMN1 lead to spinal muscular atrophy (SMA), a devastating neurodegenerative disease linked to a high rate of infant mortality. SMN2, a near identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 due to predominant skipping of SMN2 exon 7. Restoration of SMN by splicing modulation of SMN2 exon 7 or gene replacement are currently approved therapies of SMA. Human SMN genes produce a vast repertoire of circular RNAs (circRNAs). However, the mechanism of SMN circRNA generation has not yet been examined in detail. For example, it remains unknown if forward splicing impacts backsplicing that generates circRNAs containing multiple exons. Here, we employed SMN as a model system to examine the impact of intronic sequences on the generation of circRNAs. We performed our experiments in HeLa cells transiently transfected with minigenes expressing three abundantly represented circRNAs containing two or more SMN exons. We observed an enhanced rate of circRNA generation when introns joining exons to be incorporated into circRNAs were present as compared to the intronless context. These results underscore the stimulatory effect of forward splicing in the generation of circRNAs containing multiple exons. These findings are consistent with the reported low abundance of SMN circRNAs comprised of single exons. We confirmed our findings using inducible HEK 293 cells stably expressing the SMN circRNAs. Our results support the role of the exon junction complex in the generation of the exon-only-containing circRNAs. We showed that SMN circRNAs were preferentially localized in the cytoplasm. These findings provide new insights regarding our understanding of circRNA generation and open avenues to uncover novel functions of the SMN genes.
| S-EPMC9323214 | biostudies-literature
Project description:Circular RNAs (circRNAs) perform diverse functions, including the regulation of transcription, translation, peptide synthesis, macromolecular sequestration and trafficking. Inverted Alu repeats capable of forming RNA:RNA duplexes that bring splice sites together for backsplicing are known to facilitate circRNA generation. However, higher limits of circRNAs produced by a single Alu-rich gene are currently not predictable due to limitations of amplification and analyses. Here, using a tailored approach, we report a surprising diversity of exon-containing circRNAs generated by the Alu-rich Survival Motor Neuron (SMN) genes that code for SMN, an essential multifunctional protein in humans. We show that expression of the vast repertoire of SMN circRNAs is universal. Several of the identified circRNAs harbor novel exons derived from both intronic and intergenic sequences. A comparison with mouse Smn circRNAs underscored a clear impact of primate-specific Alu elements on shaping the overall repertoire of human SMN circRNAs. We show the role of DHX9, an RNA helicase, in splicing regulation of several SMN exons that are preferentially incorporated into circRNAs. Our results suggest self- and cross-regulation of biogenesis of various SMN circRNAs. These findings bring a novel perspective towards a better understanding of SMN gene function.
| S-EPMC6451121 | biostudies-literature
Project description:BackgroundStable introns and intronic fragments make up the largest population of RNA in the oocyte nucleus of the frog Xenopus tropicalis. These stable intronic sequence RNAs (sisRNAs) persist through the onset of zygotic transcription when synchronous cell division has ended, and the developing embryo consists of approximately 8000 cells. Despite their abundance, the sequence properties and biological function of sisRNAs are just beginning to be understood.ResultsTo characterize this population of non-coding RNA, we identified all of the sisRNAs in the X. tropicalis oocyte nucleus using published high-throughput RNA sequencing data. Our analysis revealed that sisRNAs, have an average length of ~?360?nt, are widely expressed from genes with multiple introns, and are derived from specific regions of introns that are GC and TG rich, while CpG poor. They are enriched in introns at both ends of transcripts but preferentially at the 3' end. The consensus binding sites of specific transcription factors such as Stat3 are enriched in sisRNAs, suggesting an association between sisRNAs and transcription factors involved in early development. Evolutionary conservation analysis of sisRNA sequences in seven vertebrate genomes indicates that sisRNAs are as conserved as other parts of introns, but much less conserved than exons.ConclusionIn total, our results indicate sisRNAs are selected intron regions with distinct properties and may play a role in gene expression regulation.
| S-EPMC7137253 | biostudies-literature
Project description:Exonic circular RNAs (circRNAs) are highly abundant RNAs generated mostly from exons of protein-coding genes. Assaying the functions of circRNAs is not straightforward as common approaches for circRNA depletion tend to also alter the levels of mRNAs generated from the hosting gene. Here we describe a methodology for specific knockdown of circRNAs in vivo with tissue and cell resolution. We also describe an experimental and computational platform for determining the potential off-target effects as well as for verifying the obtained phenotypes. Briefly, we utilize shRNAs targeted to the circRNA-specific back-splice junction to specifically downregulate the circRNA. We utilized this methodology to downregulate five circRNAs that are highly expressed in Drosophila. There were no effects on the levels of their linear counterparts or any RNA with complementarity to the expressed shRNA. Interestingly, downregulation of circCtrip resulted in developmental lethality that was recapitulated with a second shRNA. Moreover, downregulation of individual circRNAs caused specific changes in the fly head transcriptome, suggesting roles for these circRNAs in the fly nervous system. Together, our results provide a methodological approach that enables the comprehensive study of circRNAs at the organismal and cellular levels and generated for the first time flies in which specific circRNAs are downregulated.
| S-EPMC7417560 | biostudies-literature
Project description:Transfer RNAs (tRNA) are quintessential in deciphering the genetic code; disseminating nucleic acid triplets into correct amino acid identity. While this decoding function is clear, an emerging theme is that tRNA abundance and functionality can powerfully impact protein production rate, folding, activity, and messenger RNA stability. Importantly, however, the expression pattern of tRNAs is obliquely known. Here we present Quantitative Mature tRNA sequencing (QuantM-tRNA seq), a technique to monitor tRNA abundance and sequence variants secondary to RNA modifications. With QuantM-tRNA seq, we assess the tRNA transcriptome in mammalian tissues. We observe dramatic distinctions in isodecoder expression and known tRNA modifications between tissues. Remarkably, despite dramatic changes in tRNA isodecoder gene expression, the overall anticodon pool of each tRNA family is similar across tissues. These findings suggest that while anticodon pools appear to be buffered via an unknown mechanism, underlying transcriptomic and epitranscriptomic differences suggest a more complex tRNA regulatory landscape.
| S-EPMC7428014 | biostudies-literature
Project description:Circular RNAs (circRNAs) are a novel class of RNAs with closed loop structure. Blood circRNAs are widely acknowledged to be more stable than linear mRNAs, which show promising prospect to be liquid biopsy biomarkers for clinical applications. However, accumulating studies have demonstrated that sample processing delays have profound effects on blood transcriptome expression profiles, wherein knowledge remains elusive about the impacts of prolonged sample processing on blood expression profiles of circRNAs. We collected whole blood samples from three donors and isolated peripheral blood mononuclear cells (PBMCs) at six different incubation time points. We measured total RNA expression profiles using RNA sequencing (RNA-seq) and investigated the differentially expressed circRNAs, mRNAs and lncRNAs upon blood processing delay. Meanwhile, we explored the underlying inducement of aberrant expression of circRNAs against their corresponding mRNA transcripts. Finally, we utilized rMATS-turbo and CIRI-AS, respectively, to screen out differential alternative splicing (AS) events in linear mRNAs and circRNAs. Sample incubation at 4°C lasting to 48 hours (h) led to minimal effects to circRNAs' expression. However, it induced extensive alterations for mRNAs and lncRNAs when the incubation time was beyond 12 h. Additionally, only 2 h processing delays may result in profound impacts on AS events of linear mRNAs, while less impact on the equivalence of circRNAs. Our results suggested that PBMC circRNAs are stable upon sample processing delay, which are more suitable to be liquid biopsy biomarkers.
| S-EPMC9549506 | biostudies-literature
Project description:Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.
| S-EPMC6274462 | biostudies-literature
Project description:Transfer RNAs (tRNA) are quintessential in deciphering the genetic code; disseminating nucleic acid triplets into correct amino acid identity. While this decoding function is clear, an emerging theme is that tRNA abundance and functionality can powerfully impact protein production rate, folding, activity, and messenger RNA stability. Importantly, however, the expression pattern of tRNAs (in even simple systems) is obliquely known. Limited analysis suggests tRNA levels change during proliferation, differentiation, cancer, and neurodegeneration; possibly mediating changes in translation efficiency and mRNA stability. A major limitation for the field has been the ability to subject tRNA pools to high-throughput analysis as they are highly structured, modified, and of high sequence similarity. Here we present Quantitative Mature tRNA sequencing (QuantM-tRNA seq), an easily implemented high-throughput technique to monitor tRNA abundance and sequence variants (possibly due to RNA modifications). With QuantM-tRNA seq we provide a comprehensive analysis of the tRNA transcriptome from distinct mammalian tissues. We observe dramatic distinctions in isodecoder expression and likely RNA modifications between unique tissues with a particularly strong signature within the central nervous system. Remarkably, despite dramatic changes in tRNA isodecoder gene expression, the overall anticodon pool of each tRNA family is similar. These findings suggest that anticodon pools are buffered via an unknown mechanism to achieve uniform decoding throughout the body.
2020-08-04 | GSE141436 | GEO
Project description:The discovery of introns four decades ago was one of the most unexpected findings in molecular biology. Introns are sequences interrupting genes that must be removed as part of messenger RNA production. Genome sequencing projects have shown that most eukaryotic genes contain at least one intron, and frequently many. Comparison of these genomes reveals a history of long evolutionary periods during which few introns were gained, punctuated by episodes of rapid, extensive gain. However, although several detailed mechanisms for such episodic intron generation have been proposed, none has been empirically supported on a genomic scale. Here we show how short, non-autonomous DNA transposons independently generated hundreds to thousands of introns in the prasinophyte Micromonas pusilla and the pelagophyte Aureococcus anophagefferens. Each transposon carries one splice site. The other splice site is co-opted from the gene sequence that is duplicated upon transposon insertion, allowing perfect splicing out of the RNA. The distributions of sequences that can be co-opted are biased with respect to codons, and phasing of transposon-generated introns is similarly biased. These transposons insert between pre-existing nucleosomes, so that multiple nearby insertions generate nucleosome-sized intervening segments. Thus, transposon insertion and sequence co-option may explain the intron phase biases and prevalence of nucleosome-sized exons observed in eukaryotes. Overall, the two independent examples of proliferating elements illustrate a general DNA transposon mechanism that can plausibly account for episodes of rapid, extensive intron gain during eukaryotic evolution.
| S-EPMC5684705 | biostudies-literature
Project description:Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) cause ?2% of all human cancers. RNase R-resistant RNA sequencing revealed that both gammaherpesviruses encode multiple, uniquely stable, circular RNAs (circRNA). EBV abundantly expressed both exon-only and exon-intron circRNAs from the BamHI A rightward transcript (BART) locus (circBARTs) formed from a spliced BART transcript and excluding the EBV miRNA region. The circBARTs were expressed in all verified EBV latency types, including EBV-positive posttransplant lymphoproliferative disease, Burkitt lymphoma, nasopharyngeal carcinoma, and AIDS-associated lymphoma tissues and cell lines. Only cells infected with the B95-8 EBV strain, with a 12-kb BART locus deletion, were negative for EBV circBARTs. Less abundant levels of EBV circRNAs originating from LMP2- and BHLF1-encoding genes were also identified. The circRNA sequencing of KSHV-infected primary effusion lymphoma cells revealed a KSHV-encoded circRNA from the vIRF4 locus (circvIRF4) that was constitutively expressed. In addition, KSHV polyadenylated nuclear (PAN) RNA locus generated a swarm (>100) of multiply backspliced, low-abundance RNase R-resistant circRNAs originating in both sense and antisense directions consistent with a novel hyperbacksplicing mechanism. In EBV and KSHV coinfected cells, exon-only EBV circBARTs were located more in the cytoplasm, whereas the intron-retaining circBARTs were found in the nuclear fraction. KSHV circvIRF4 and circPANs were detected in both nuclear and cytoplasmic fractions. Among viral circRNAs tested, none were found in polysome fractions from KSHV-EBV coinfected BC1 cells, although low-abundance protein translation from viral circRNAs could not be excluded. The circRNAs are a new class of viral transcripts expressed in gammaherpesvirus-related tumors that might contribute to viral oncogenesis.
| S-EPMC6140489 | biostudies-literature