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The identification of a new subclass of circular RNAs that are predominantly nuclear and promote transcription of their parental genes reveals a new regulatory function for these noncoding RNAs. Noncoding RNAs (ncRNAs) have numerous roles in development and disease, and one of the prominent roles is to regulate gene expression. A vast number of circular RNAs (circRNAs) have been identified, and some have been shown to function as microRNA sponges in animal cells. Here, we report a class of circRNAs associated with RNA polymerase II in human cells. In these circRNAs, exons are circularized with introns 'retained' between exons; we term them exon-intron circRNAs or EIciRNAs. EIciRNAs predominantly localize in the nucleus, interact with U1 snRNP and promote transcription of their parental genes. Our findings reveal a new role for circRNAs in regulating gene expression in the nucleus, in which EIciRNAs enhance the expression of their parental genes in cis , and highlight a regulatory strategy for transcriptional control via specific RNA-RNA interaction between U1 snRNA and EIciRNAs.

Repetitive elements (REs) comprise 40-60% of the mammalian genome and have been shown to epigenetically influence the expression of genes through the formation of fusion transcript (FTs). We previously showed that an intracisternal A particle forms an FT with the agouti gene in mice, causing obesity/type 2 diabetes. To determine the frequency of FTs genome-wide, we developed a TopHat-Fusion-based analytical pipeline to identify FTs with high specificity. We applied it to an RNA-seq dataset from the nucleus accumbens (NAc) of mice repeatedly exposed to cocaine. Cocaine was previously shown to increase the expression of certain REs in this brain region. Using this pipeline that can be applied to single- or paired-end reads, we identified 438 genes expressing 813 different FTs in the NAc. Although all types of studied repeats were present in FTs, simple sequence repeats were underrepresented. Most importantly, reverse-transcription and quantitative PCR validated the expression of selected FTs in an independent cohort of animals, which also revealed that some FTs are the prominent isoforms expressed in the NAc by some genes. In other RNA-seq datasets, developmental expression as well as tissue specificity of some FTs differed from their corresponding non-fusion counterparts. Finally, in silico analysis predicted changes in the structure of proteins encoded by some FTs, potentially resulting in gain or loss of function. Collectively, these results indicate the robustness of our pipeline in detecting these new isoforms of genes, which we believe provides a valuable tool to aid in better understanding the broad role of REs in mammalian cellular biology.

Many viruses use overprinting (alternate reading frame utilization) as a means to increase protein diversity in genomes severely constrained by size. However, the evolutionary steps that facilitate the de novo generation of a novel protein within an ancestral ORF have remained poorly characterized. Here, we describe the identification of an overprinting gene, expressed from an Alternate frame of the Large T Open reading frame (ALTO) in the early region of Merkel cell polyomavirus (MCPyV), the causative agent of most Merkel cell carcinomas. ALTO is expressed during, but not required for, replication of the MCPyV genome. Phylogenetic analysis reveals that ALTO is evolutionarily related to the middle T antigen of murine polyomavirus despite almost no sequence similarity. ALTO/MT arose de novo by overprinting of the second exon of T antigen in the common ancestor of a large clade of mammalian polyomaviruses. Taking advantage of the low evolutionary divergence and diverse sampling of polyomaviruses, we propose evolutionary transitions that likely gave birth to this protein. We suggest that two highly constrained regions of the large T antigen ORF provided a start codon and C-terminal hydrophobic motif necessary for cellular localization of ALTO. These two key features, together with stochastic erasure of intervening stop codons, resulted in a unique protein-coding capacity that has been preserved ever since its birth. Our study not only reveals a previously undefined protein encoded by several polyomaviruses including MCPyV, but also provides insight into de novo protein evolution.

Recent studies have provided strong evidence for a regulatory link among chromatin structure, histone modification, and splicing regulation. However, it is largely unknown how local histone modification patterns surrounding alternative exons are connected to differential alternative splicing outcomes. Here we show that splicing regulator Hu proteins can induce local histone hyperacetylation by association with their target sequences on the pre-mRNA surrounding alternative exons of two different genes. In both primary and mouse embryonic stem cell-derived neurons, histone hyperacetylation leads to an increased local transcriptional elongation rate and decreased inclusion of these exons. Furthermore, we demonstrate that Hu proteins interact with histone deacetylase 2 and inhibit its deacetylation activity. We propose that splicing regulators may actively modulate chromatin structure when recruited to their target RNA sequences cotranscriptionally. This \"reaching back\" interaction with chromatin provides a means to ensure accurate and efficient regulation of alternative splicing.

Alternative splicing of pre-messenger RNA (mRNA) is a critical stage of gene regulation in response to environmental stimuli. Here we show that DAZAP1, an RNA-binding protein involved in mammalian development and spermatogenesis, promotes inclusion of weak exons through specific recognition of diverse cis -elements. The carboxy-terminal proline-rich domain of DAZAP1 interacts with and neutralizes general splicing inhibitors, and is sufficient to activate splicing when recruited to pre-mRNA. This domain is phosphorylated by the MEK/Erk (extracellular signal-regulated protein kinase) pathway and this modification is essential for the splicing regulatory activity and the nuclear/cytoplasmic translocation of DAZAP1. Using mRNA-seq, we identify endogenous splicing events regulated by DAZAP1, many of which are involved in maintaining cell growth. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect. Taken together, these studies reveal a molecular mechanism that integrates splicing control into MEK/Erk-regulated cell proliferation. DAZAP1 is a multi-functional RNA-binding protein that affects diverse aspects of RNA metabolism. Here, Choudhury et al. demonstrate that DAZAP1 acts as a general splice activator regulated by MEK/ERK signalling and thereby serves to integrate splice control into the regulation of cellular proliferation.

Immediate early genes are rapidly transcribed in response to neuronal activity, but the underlying mechanism is unclear. The authors show that this rapid transcription is mediated by a stalled RNA polymerase II, poised just downstream of the transcription start site. RNAi-depletion of negative elongation factor compromises the rapid transcription. Transcription of immediate early genes (IEGs) in neurons is highly sensitive to neuronal activity, but the mechanism underlying these early transcription events is largely unknown. We found that several IEGs, such as Arc (also known as Arg3.1 ), are poised for near-instantaneous transcription by the stalling of RNA polymerase II (Pol II) just downstream of the transcription start site in rat neurons. Depletion through RNA interference of negative elongation factor, a mediator of Pol II stalling, reduced the Pol II occupancy of the Arc promoter and compromised the rapid induction of Arc and other IEGs. In contrast, reduction of Pol II stalling did not prevent transcription of IEGs that were expressed later and largely lacked promoter-proximal Pol II stalling. Together, our data strongly indicate that the rapid induction of neuronal IEGs requires poised Pol II and suggest a role for this mechanism in a wide variety of transcription-dependent processes, including learning and memory.

In addition to coding information, human exons contain sequences necessary for correct splicing. These elements are known to be under purifying selection and their disruption can cause disease. However, the density of functional exonic splicing information remains profoundly uncertain. Several groups have experimentally investigated how mutations at different exonic positions affect splicing. They have found splice information to be distributed widely in exons, with one estimate putting the proportion of splicing-relevant nucleotides at >90%. These results suggest that splicing could place a major pressure on exon evolution. However, analyses of sequence conservation have concluded that the need to preserve splice regulatory signals only slightly constrains exon evolution, with a resulting decrease in the average human rate of synonymous evolution of only 1–4%. Why do these two lines of research come to such different conclusions? Among other reasons, we suggest that the methods are measuring different things: one assays the density of sites that affect splicing, the other the density of sites whose effects on splicing are visible to selection. In addition, the experimental methods typically consider short exons, thereby enriching for nucleotides close to the splice junction, such sites being enriched for splice-control elements. By contrast, in part owing to correction for nucleotide composition biases and to the assumption that constraint only operates on exon ends, the conservation-based methods can be overly conservative.

Background Most eukaryotic genes produce different transcripts of multiple isoforms by inclusion or exclusion of particular exons. The isoforms of a gene often play diverse functional roles, and thus it is necessary to accurately measure isoform expressions as well as gene expressions. While previous studies have demonstrated the strong agreement between mRNA sequencing (RNA-seq) and array-based gene and/or isoform quantification platforms (Microarray gene expression and Exon-array), the more recently developed NanoString platform has not been systematically evaluated and compared, especially in large-scale studies across different cancer domains. Results In this paper, we present a large-scale comparative study among RNA-seq, NanoString, array-based, and RT-qPCR platforms using 46 cancer cell lines across different cancer types. The goal is to understand and evaluate the calibers of the platforms for measuring gene and isoform expressions in cancer studies. We first performed NanoString experiments on 59 cancer cell lines with 404 custom-designed probes for measuring the expressions of 478 isoforms in 155 genes, and additional RT-qPCR experiments for a subset of the measured isoforms in 13 cell lines. We then combined the data with the matched RNA-seq, Exon-array, and Microarray data of 46 of the 59 cell lines for the comparative analysis. Conclusion In the comparisons of the platforms for measuring the expressions at both isoform and gene levels, we found that (1) the agreement on isoform expressions is lower than the agreement on gene expressions across the four platforms; (2) NanoString and Exon-array are not consistent on isoform quantification even though both techniques are based on hybridization reactions; (3) RT-qPCR experiments are more consistent with RNA-seq and Exon-array than NanoString in isoform quantification; (4) different RNA-seq isoform quantification methods show varying estimation results, and among the methods, Net-RSTQ and eXpress are more consistent across the platforms; and (5) RNA-seq has the best overall consistency with the other platforms on gene expression quantification.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it is requisite to rigorously optimise oligonucleotide chemistries as well as chemical modifications of ASOs. To achieve this, here, we aim to develop a novel enhanced green fluorescence protein (EGFP)-based reporter assay system that allows us to perform efficient and high-throughput screenings for ASOs. We design a new expression vector with a CAG promoter to detect the EGFP fluorescence only when skipping of mdx -type exon 23 is induced by ASOs. Then, an accurate screening was successfully conducted in C57BL/6 primary myotubes using phosphorodiamidate morpholino oligomer or locked nucleic acids (LNA)/2′-OMe mixmers with different extent of LNA inclusion. We accordingly generated a novel transgenic mouse model with this EGFP expression vector (EGFP- mdx 23 Tg). Finally, we confirmed that the EGFP- mdx 23 Tg provided a highly sensitive platform to check the effectiveness as well as the biodistribution of ASOs for exon skipping therapy. Thus, the assay system provides a simple yet highly sensitive platform to optimise oligonucleotide chemistries as well as chemical modifications of ASOs.

Epithelial-mesenchymal interactions are crucial for the development of numerous animal structures. Thus, unraveling how molecular tools are recruited in different lineages to control interplays between these tissues is key to understanding morphogenetic evolution. Here, we study Esrp genes, which regulate extensive splicing programs and are essential for mammalian organogenesis. We find that Esrp homologs have been independently recruited for the development of multiple structures across deuterostomes. Although Esrp is involved in a wide variety of ontogenetic processes, our results suggest ancient roles in non-neural ectoderm and regulating specific mesenchymal-to-epithelial transitions in deuterostome ancestors. However, consistent with the extensive rewiring of Esrp -dependent splicing programs between phyla, most developmental defects observed in vertebrate mutants are related to other types of morphogenetic processes. This is likely connected to the origin of an event in Fgfr , which was recruited as an Esrp target in stem chordates and subsequently co-opted into the development of many novel traits in vertebrates. Epithelial-mesenchymal interplays are essential to many ontogenetic processes in vertebrates. Here Burguera et al . show diverse embryonic morphogenetic processes regulated by Epithelial Splicing Regulatory Protein ( Esrp ) in different deuterostome species.