A survey of RNA secondary structural propensity encoded within human herpesvirus genomes: global comparisons and local motifs

There are nine herpesviruses known to infect humans, of which Epstein–Barr virus (EBV) is the most widely distributed (>90% of adults infected). This ubiquitous virus is implicated in a variety of cancers and autoimmune diseases. Previous analyses of the EBV genome revealed numerous regions with evidence of generating unusually stable and conserved RNA secondary structures and led to the discovery of a novel class of EBV non-coding (nc)RNAs: the stable intronic sequence (sis)RNAs. To gain a better understanding of the roles of RNA structure in EBV biology and pathogenicity, we revisit EBV using recently developed tools for genome-wide motif discovery and RNA structural characterization. This corroborated previous results and revealed novel motifs with potential functionality; one of which has been experimentally validated. Additionally, since many herpesviruses increasingly rival the seroprevalence of EBV (VZV, HHV-6 and HHV-7 being the most notable), analyses were expanded to include all sequenced human Herpesvirus RefSeq genomes, allowing for genomic comparisons. In total 10 genomes were analyzed, for EBV (types 1 and 2), HCMV, HHV-6A, HHV-6B, HHV-7, HSV-1, HSV-2, KSHV, and VZV. All resulting data were archived in the RNAStructuromeDB (https://structurome.bb.iastate.edu/herpesvirus) to make them available to a wide array of researchers.

In-Depth Analysis Reveals Production of Circular RNAs from Non-Coding Sequences

The sequencing of total RNA depleted for ribosomal sequences remains the method of choice for the study of circRNAs. Our objective was to characterize non-canonical circRNAs, namely not originating from back splicing and circRNA produced by non-coding genes. To this end, we analyzed a dataset from porcine testis known to contain about 100 intron-derived circRNAs. Labelling reads containing a circular junction and originating from back splicing provided information on the very small contribution of long non-coding genes to the production of canonical circRNAs. Analyses of the other reads revealed two origins for non-canonical circRNAs: (1) Intronic sequences for lariat-derived intronic circRNAs and intron circles, (2) Mono-exonic genes (mostly non-coding) for either a new type of circRNA (including only part of the exon: sub-exonic circRNAs) or, even more rarely, mono-exonic canonical circRNAs. The most complex set of sub-exonic circRNAs was produced by RNase_MRP (ribozyme RNA). We specifically investigated the intronic circRNA of ATXN2L, which is probably an independently transcribed sisRNA (stable intronic sequence RNA). We may be witnessing the emergence of a new non-coding gene in the porcine genome. Our results are evidence that most non-canonical circRNAs originate from non-coding sequences.

A deep intronic variant activates a pseudoexon in the MTM1 gene in a family with X-linked myotubular myopathy

We report a novel intronic variant in the MTM1 gene in four males in a family with severe X-linked myotubular myopathy. The A>G variant in deep intronic space activates a cryptic 5’ donor splice site resulting in the inclusion of a 48bp pseudoexon into the mature MTM1 mRNA. The variant is present in all affected males, absent in unaffected males and heterozygous in the mother of the affected males. The included intronic sequence contains a premature stop codon and experiments using a translational inhibitor indicate that the mutant mRNAs undergo nonsense-mediate decay. We conclude that affected males produce no, or low, levels of myotubularin-1 protein leading to a severe neonatal myopathy. The study highlights the need to consider non-coding variants in genomic screening in families with X-linked myotubular myopathy.

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable 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. Results: To characterize this population of noncoding 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 bps, 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. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, and may play a role in gene expression regulation .

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable 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. Results: To characterize this population of noncoding 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 bps, 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. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, and may play a role in gene expression regulation.

Stable intronic sequence RNAs (sisRNAs) are selected regions in introns with distinct properties

Background: Stable 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. Results: To characterize this population of noncoding 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 bps, 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. Conclusion: In total, our results indicate sisRNAs are selected intron regions with distinct properties, supporting a biological function in gene expression regulation.

Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy

Duchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/frameshift mutations in DMD and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in DMD in the Japanese Registry of Muscular Dystrophy. For each DMD mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in DMD gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in DMD and for interpreting the phenotypes of the BMD patients.