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Comprehensive analysis of translation from overexpressed circular RNAs reveals pervasive translation from linear transcripts.

ABSTRACT: Circular RNAs (circRNAs) encompass a widespread and conserved class of RNAs, which are generated by back-splicing of downstream 5' to upstream 3' splice sites. CircRNAs are tissue-specific and have been implicated in diseases including cancer. They can function as sponges for microRNAs (miRNAs) or RNA binding proteins (RBPs), for example. Moreover, some contain open reading frames (ORFs) and might be translated. The functional relevance of such peptides, however, remains largely elusive. Here, we report that the ORF of circZNF609 is efficiently translated when expressed from a circZNF609 overexpression construct. However, endogenous proteins could not be detected. Moreover, initiation of circZNF609 translation is independent of m6A-generating enzyme METTL3 or RNA sequence elements such as internal ribosome entry sites (IRESs). Surprisingly, a comprehensive mutational analysis revealed that deletion constructs, which are deficient in producing circZNF609, still generate the observed protein products. This suggests that the apparent circZNF609 translation originates from trans-splicing by-products of the overexpression plasmids and underline that circRNA overexpression constructs need to be evaluated carefully, particularly when functional studies are performed.

SUBMITTER: Ho-Xuan H

PROVIDER: S-EPMC7544230 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Comprehensive analysis of translation from overexpressed circular RNAs reveals pervasive translation from linear transcripts.

Ho-Xuan Hung H Glažar Petar P Latini Claudia C Heizler Kevin K Haase Jacob J Hett Robert R Anders Marvin M Weichmann Franziska F Bruckmann Astrid A Van den Berg Debbie D Hüttelmaier Stefan S Rajewsky Nikolaus N Hackl Christina C Meister Gunter G

Nucleic acids research 20201001 18

Circular RNAs (circRNAs) encompass a widespread and conserved class of RNAs, which are generated by back-splicing of downstream 5' to upstream 3' splice sites. CircRNAs are tissue-specific and have been implicated in diseases including cancer. They can function as sponges for microRNAs (miRNAs) or RNA binding proteins (RBPs), for example. Moreover, some contain open reading frames (ORFs) and might be translated. The functional relevance of such peptides, however, remains largely elusive. Here, w ...[more]

PMID: 32955563

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Project description:BackgroundNon-co-linear (NCL) transcripts consist of exonic sequences that are topologically inconsistent with the reference genome in an intragenic fashion (circular or intragenic trans-spliced RNAs) or in an intergenic fashion (fusion or intergenic trans-spliced RNAs). On the basis of RNA-seq data, numerous NCL event detectors have been developed and detected thousands of NCL events in diverse species. However, there are great discrepancies in the identification results among detectors, indicating a considerable proportion of false positives in the detected NCL events. Although several helpful guidelines for evaluating the performance of NCL event detectors have been provided, a systematic guideline for measurement of NCL events identified by existing tools has not been available.ResultsWe develop a software, NCLcomparator, for systematically post-screening the intragenic or intergenic NCL events identified by various NCL detectors. NCLcomparator first examine whether the input NCL events are potentially false positives derived from ambiguous alignments (i.e., the NCL events have an alternative co-linear explanation or multiple matches against the reference genome). To evaluate the reliability of the identified NCL events, we define the NCL score (NCLscore) based on the variation in the number of supporting NCL junction reads identified by the tools examined. Of the input NCL events, we show that the ambiguous alignment-derived events have relatively lower NCLscore values than the other events, indicating that an NCL event with a higher NCLscore has a higher level of reliability. To help selecting highly expressed NCL events, NCLcomparator also provides a series of useful measurements such as the expression levels of the detected NCL events and their corresponding host genes and the junction usage of the co-linear splice junctions at both NCL donor and acceptor sites.ConclusionNCLcomparator provides useful guidelines, with the input of identified NCL events from various detectors and the corresponding paired-end RNA-seq data only, to help users selecting potentially high-confidence NCL events for further functional investigation. The software thus helps to facilitate future studies into NCL events, shedding light on the fundamental biology of this important but understudied class of transcripts. NCLcomparator is freely accessible at https://github.com/TreesLab/NCLcomparator .

Project description:Human cytomegalovirus (HCMV) infects a large portion of the human population globally. Several HCMV-derived noncoding RNAs are involved in the regulation of viral gene expression and the virus life cycle. Here, we reported that circRNAs are a new class of HCMV transcripts. We bioinformatically predict 704 candidate circRNAs encoded by the TB40/E strain and 230 encoded by the HAN strain. We also systematically compare circRNA features, including the breakpoint sequence consensus, strand preference, length distribution, and exon numbers between host genome-encoded circRNAs and viral circRNAs, and showed that the unique characteristics of viral circRNAs are correlated with their genome types. Furthermore, we experimentally confirmed 324 back-splice junctions (BSJs) from three HCMV strains, Towne, TB40/E, and Toledo, and identified 4 representative HCMV circRNAs by RNase R treatment. Interestingly, we also showed that HCMV contains alternative back-splicing circRNAs. We developed a new amplified FISH method that allowed us to visualize circRNAs and quantify the number of circRNA molecules in the infected cells. The competitive endogenous RNA network analysis suggests that HCMV circRNAs play important roles in viral DNA synthesis via circRNA-miRNA-mRNA networks. Our findings highlight that circRNAs are an important component of the HCMV transcriptome that may contribute to viral replication and pathogenesis. IMPORTANCE HCMV infects 40% to 100% of the human population globally and may be a life-threatening pathogen in immunocompromised individuals. CircRNA is a family of unique RNA that is the most newly found and remains unknown in many aspects. Our current studies computationally identified HCMV-encoded circRNAs and confirmed the existence of the HCMV circRNAs in the infected cells. We systematically compared the features between host and different viral circRNAs and found that the unique characteristics of circRNAs were correlated with their genome types. We also first reported that HCMV contained alternative back-splicing circRNAs. More importantly, we developed a new amplified FISH method which allowed us for the first time not only to visualize circRNAs but also to quantify the number of circRNA molecules in the infected cells. This work describes a novel component of HCMV transcriptome bringing a new understanding of HCMV biology and disease.

Dataset Information

Comprehensive analysis of translation from overexpressed circular RNAs reveals pervasive translation from linear transcripts.

Publications

Comprehensive analysis of translation from overexpressed circular RNAs reveals pervasive translation from linear transcripts.

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