Project description:We performed Ribosome-protected mRNA fragment sequencing (Ribo-Seq) to determine if retinal circular RNAs are undergoing translation
Project description:Recently, it has been demonstrated that genomes of many species express single stranded RNAs with covalently closed ends, named circular RNAs. Their regulatory potential and functional relevance are just starting to be revealed. Here we present a novel computational tool, seekCRIT (seek for differentially expressed Circular RNAs In Transcriptome), that identifies circular RNAs and detects their differential expression between two conditions. Using seekCRIT we identified the circular RNAs that are expressed in the neural retina and determined that the majority of them (74%) are expressed in both, ischemic and normal conditions. We identified over 40 circular RNAs that were differentially expressed between both conditions and validated these experimentally using qRT-PCR. The high validation rate of 90% with a false discovery rate (FDR) of < 5% demonstrates the accuracy and reliability of seekCRIT.
Project description:Circular RNAs (circRNAs), formed by the atypical head-to-tail splicing of exons, have re-emerged as a potentially interesting RNA species given recent reports of a surprising diversity and abundance of circRNA in organisms ranging from worm to human. Here, using deep RNA sequencing, we profiled different RNA species in mouse and observed that circRNAs are significantly enriched in neural tissue, relative to other tissues. Using PacBio sequencing, we determined, for the first time, the circular structure of this population of circRNAs as well as their full-length sequences. We discovered that a disproportionate fraction of the brain circRNA population is derived from host genes that code for synaptic proteins. Moreover, based on the separate profiling of the RNAs localized in neuronal cell bodies and neuropil (enriched in axons and dendrites), we found that, on average, circular RNAs are more enriched in the neuropil than their host gene mRNA isoforms. Using high resolution in situ hybridization we, for the first time, directly visualized circRNA punctae in the dendrites of neurons. The host gene origin and location of the circRNA in neurons suggest the possibility that circRNAs might participate in the regulation of synaptic function and plasticity. Consistent with this idea, we observed via profiling at different developmental stages, that the abundance of many circular RNAs changes abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibit significant up or down-regulation. These data indicate that brain circRNAs are positioned to respond to and regulate synaptic function. Circular RNA profiling in 13 different samples in mice and four samples in rat, using Illumina sequencing
Project description:Circular RNAs (circRNAs) are a class of abundant RNAs with ambiguous function. Although some circRNAs can be translated through IRES driven mechanisms, the scope and functions of circRNA translation are unclear because endogenous IRESs are rare. To determine the prevalence and mechanism of circRNA translation, we developed a cell-based system to screen random sequences and identified 97 overrepresented AU-rich hexamers (>2% of all hexamers) that drive cap-independent translation of circRNAs. These IRES-like short elements are significantly enriched in circRNAs and sufficient to drive circRNA translation. We further identified multiple trans-acting factors that bind these IRES-like short elements to initiate translation. Using mass-spectrometry data, hundreds of circRNA-coded peptides were identified, most of which have low abundance due to rapid degradation. As judged by mass-spectrometry, 50% of translatable endogenous circRNAs undergo rolling circle translation, several of which were experimentally validated by western blotting. Consistently, the mutation of the IRES-like short element in one circRNA reduced its translation. Collectively, our findings suggest a pervasive translation of circRNAs, providing profound implications in circRNA function.
Project description:The human neural retina is enriched for alternative splicing, and it is estimated that more than 10% of variants associated with inherited retinal diseases (IRDs) alter splicing. Previous research mainly used short-read RNA-sequencing techniques to investigate retina-specific splicing and splicing factors. However, this technique provides limited information about transcript isoforms. To gain a deeper understanding of the human neural retina and its isoforms, we generated a proteogenomic atlas that combined PacBio long-read RNA-sequencing data with mass-spectrometry and whole-genome sequencing data from three healthy human neural retina samples. RNA-sequencing revealed that one-third of all transcripts were novel, and for IRD-associated genes, even 43% were novel. The most common novel elements of these transcripts were alternative poly(A) sites, exon elongation, and intron retention. Some novel elements affect the non-coding region but for more than 50% of the novel transcripts a novel open reading frame was predicted. Using proteomics, ten novel peptides confirmed novel isoforms in five genes. Additionally, we found novel isoforms of IMPDH1, an IRD-associated gene, with supporting peptide evidence. This study provides a comprehensive overview of the transcript and protein isoforms expressed in the healthy human neural retina. Moreover, it highlights the importance of studying tissue specific transcriptomes in greater detail to better understand tissue-specific regulation and to identify disease-causing variants.
Project description:The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs requires internal ribosome entry sites (IRES) if they are to undergo translation without 5’ cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify over 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue -specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is down regulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease.
Project description:Circular RNAs exhibit limited evidence for translation, or translation regulation of the mRNA-counterpart in terminal hematopoiesis
Project description:Circular RNAs (circRNAs), formed by the atypical head-to-tail splicing of exons, have re-emerged as a potentially interesting RNA species given recent reports of a surprising diversity and abundance of circRNA in organisms ranging from worm to human. Here, using deep RNA sequencing, we profiled different RNA species in mouse and observed that circRNAs are significantly enriched in neural tissue, relative to other tissues. Using PacBio sequencing, we determined, for the first time, the circular structure of this population of circRNAs as well as their full-length sequences. We discovered that a disproportionate fraction of the brain circRNA population is derived from host genes that code for synaptic proteins. Moreover, based on the separate profiling of the RNAs localized in neuronal cell bodies and neuropil (enriched in axons and dendrites), we found that, on average, circular RNAs are more enriched in the neuropil than their host gene mRNA isoforms. Using high resolution in situ hybridization we, for the first time, directly visualized circRNA punctae in the dendrites of neurons. The host gene origin and location of the circRNA in neurons suggest the possibility that circRNAs might participate in the regulation of synaptic function and plasticity. Consistent with this idea, we observed via profiling at different developmental stages, that the abundance of many circular RNAs changes abruptly at a time corresponding to synaptogenesis. In addition, following a homeostatic downscaling of neuronal activity many circRNAs exhibit significant up or down-regulation. These data indicate that brain circRNAs are positioned to respond to and regulate synaptic function.