scholarly journals An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys

2018 ◽  
Author(s):  
Marilyn Parra ◽  
Ben W. Booth ◽  
Richard Weiszmann ◽  
Brian Yee ◽  
Gene W. Yeo ◽  
...  
Keyword(s):  
Intron Retention ◽  
Developmental Regulation ◽  
Splice Junction ◽  
Rna Seq ◽  
Nonsense Mediated Decay ◽  
Genome Wide ◽  
Reporter Assays ◽  
Retained Introns ◽  
Splice Junctions ◽  
Intron 4

AbstractDuring terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the SF3B1 splicing factor gene, which expresses ~50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron. Minigene splicing reporter assays showed that these cassettes promote IR. Genome-wide analysis of splice junction reads demonstrated that cryptic noncoding cassettes are much more common in large (>1kb) retained introns than they are in small retained introns or in non-retained introns. Functional assays showed that heterologous cassettes can promote retention of intron 4 in the SF3B1 splicing reporter. Although many of these cryptic exons were spliced inefficiently, they exhibited substantial binding of U2AF1 and U2AF2 adjacent to their splice acceptor sites. We propose that these exons function as decoys that engage the intron-terminal splice sites, blocking cross-intron interactions required for excision. Developmental regulation of decoy function underlies a major component of the erythroblast IR program.

scholarly journals iREAD: A Tool for Intron Retention Detection from RNA-seq Data

2017 ◽  
Author(s):  
Hong-Dong Li ◽  
Cory C. Funk ◽  
Nathan D. Price
Keyword(s):  
Intron Retention ◽  
Distribution Patterns ◽  
Functional Enrichment ◽  
Command Line ◽  
Rna Seq ◽  
Text File ◽  
Command Line Interface ◽  
Genome Wide ◽  
A Genome ◽  
Retained Introns

AbstractSummaryDetecting intron retention (IR) events is emerging as a specialized need for RNA-seq data analysis. Here we present iREAD (intron REtention Analysis and Detector), a tool to detect IR events genome-wide from high-throughput RNA-seq data. The command line interface for iREAD is implemented in Python. iREAD takes as input an existing BAM file, representing the transcriptome, and a text file containing the intron coordinates of a genome. It then 1) counts all reads that overlap intron regions, 2) detects IR vents by analyzing features of reads such as depth and distribution patterns, and 3) outputs a list of retained introns into a tab-delimited text file. The output can be directly used for further exploratory analysis such as differential intron expression and functional enrichment. iREAD provides a new and generic tool to interrogate poly-A enriched transcriptomic data of intron regions.Availabilitywww.libpls.net/[email protected]

scholarly journals SF3B1 Gene Expression in Erythroid Cells Is Regulated By Intron Retention Via a Posttranscriptional Mechanism Involving Cryptic Exons Proposed to Function As Splicing Decoys

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 931-931
Author(s):  
Marilyn Parra ◽  
Benjamin W Booth ◽  
Gene W Yeo ◽  
Richard Weiszmann ◽  
Susan E Celniker ◽  
...  
Keyword(s):  
Rna Binding ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Intron Retention ◽  
K562 Cells ◽  
Rna Seq ◽  
Splice Sites ◽  
Site Factors ◽  
Splice Junctions ◽  
Intron 4

Abstract Proper expression of the MDS-disease gene, SF3B1, ensures appropriate pre-mRNA splicing in erythroid progenitors and during terminal erythropoiesis. We previously showed that the SF3B1 gene is post-transcriptionally regulated in a differentiation stage-specific manner by intron retention (IR), such that ~50% of its transcripts in mature erythroblasts retain intron 4. Based on new mechanistic studies, we propose a model in which mostly unannotated and noncoding exons within intron 4 function as splicing decoys; i.e., they promote retention of intron 4 by interacting with, and blocking splice sites of, the adjacent exons 4 and 5. A total of six putative decoy exons were revealed via RT-PCR and RNA-seq analysis of RNA from erythroblasts treated with inhibitors of nonsense-mediated decay. That decoy exons have IR-promoting activity is suggested by several criteria. First, the frequency of interaction between constitutive exons 4 and 5 and putative decoy exons within intron 4, measured by the abundance of splice junctions in RNA-seq read data, is temporally correlated with levels of intron 4 retention during terminal erythropoiesis. Both IR and decoy splice junctions were low in early stage erythroblasts and much higher in mature erythroblasts. Second, selected decoy exons exhibited IR-promoting activity in the context of minigene splicing reporters expressing the exon 3-6 region of SF3B1 in transfected K562 cells. The wild type minigene reproduced the intron-specific retention phenotype, since it was fully spliced at introns 3 and 5 but exhibited substantial retention of intron 4, whereas deletion of decoy exon 4e, or mutation of its splice sites, substantially decreased IR. Third, RBP (RNA binding protein) cross-linking data from K562 cells show that 3' splice site factors including U2AF1 and U2AF2 can bind specifically to 3' splice sites of intron 4's decoy exons. Finally, several experiments showed that IR-promoting activity of decoy exons is a more general phenomenon that likely governs IR in other erythroid genes. We observed not only that SF3B1 intron 4 decoy exons could promote IR in heterologous contexts, but also that predicted decoy exons from other erythroblast transcripts could promote IR in the SF3B1 minigene. Apart from this experimental data, comparative genomics revealed that the SF3B1 decoy exons are extremely conserved among vertebrate genomes, with two of the exons being essentially identical from fish to humans. Together this data supports the hypothesis that a subset of up-regulated IR events in late erythroblasts are controlled by decoy exons that block productive splicing at the flanking exons. We propose that regulated IR is an important post-transcriptional mechanism for adjusting cellular splicing capacity during terminal erythropoiesis by regulating expression of key splicing factors such as SF3B1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays

2018 ◽  
Vol 28 (12) ◽  
pp. 1826-1840 ◽  
Author(s):  
Steffen Erkelenz ◽  
Stephan Theiss ◽  
Wolfgang Kaisers ◽  
Johannes Ptok ◽  
Lara Walotka ◽  
...  
Keyword(s):  
Splice Site ◽  
Rna Seq ◽  
Genome Wide ◽  
Reporter Assays ◽  
Splice Site Usage

scholarly journals Regulation of DNA methylation on key parasitism genes of Cysticercus cellulosae revealed by integrative epigenomic-transcriptomic analyses

Hereditas ◽  
2021 ◽  
Vol 158 (1) ◽  
Author(s):  
Xinrui Wang ◽  
Weiyi Song ◽  
Guanyu Ji ◽  
Yining Song ◽  
Xiaolei Liu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Developmental Regulation ◽  
Taenia Solium ◽  
Rna Seq ◽  
Host Interactions ◽  
Genome Wide ◽  
Cysticercus Cellulosae ◽  
Genes Encoding ◽  
Genome Bisulfite Sequencing ◽  
Parasitism Genes

Abstract Background The life cycle of Taenia solium is characterized by different stages of development, requiring various kinds of hosts that can appropriately harbor the eggs (proglottids), the oncospheres, the larvae and the adults. Similar to other metazoan pathogens, T. solium undergoes transcriptional and developmental regulation via epigenetics during its complex lifecycle and host interactions. Result In the present study, we integrated whole-genome bisulfite sequencing and RNA-seq technologies to characterize the genome-wide DNA methylation and its effect on transcription of Cysticercus cellulosae of T. solium. We confirm that the T. solium genome in the cysticercus stage is epigenetically modified by DNA methylation in a pattern similar to that of other invertebrate genomes, i.e., sparsely or moderately methylated. We also observed an enrichment of non-CpG methylation in defined genetic elements of the T. solium genome. Furthermore, an integrative analysis of both the transcriptome and the DNA methylome indicated a strong correlation between these two datasets, suggesting that gene expression might be tightly regulated by DNA methylation. Importantly, our data suggested that DNA methylation might play an important role in repressing key parasitism-related genes, including genes encoding excretion-secretion proteins, thereby raising the possibility of targeting DNA methylation processes as a useful strategy in therapeutics of cysticercosis.

scholarly journals Workflow for Genome-Wide Determination of Pre-mRNA Splicing Efficiency from Yeast RNA-seq Data

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Martin Převorovský ◽  
Martina Hálová ◽  
Kateřina Abrhámová ◽  
Jiří Libus ◽  
Petr Folk
Keyword(s):  
Growth Conditions ◽  
Mrna Splicing ◽  
Rna Seq ◽  
Fastq Format ◽  
Genome Wide ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Splicing Efficiency ◽  
Splice Junctions

Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5′ and 3′ splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.

scholarly journals Interrogation of genome-wide, experimentally dissected gene regulatory networks reveals mechanisms underlying dynamic cellular state control

2021 ◽  
Author(s):  
Xiangtian Tan ◽  
Jeremy Worley ◽  
Mikko Turunen ◽  
Kelly Wong ◽  
Ester Calvo Fernandez ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Regulatory Protein ◽  
State Control ◽  
Rna Seq ◽  
Protein Activity ◽  
Genome Wide ◽  
Adenocarcinoma Cells ◽  
Reporter Assays ◽  
Gene Regulatory ◽  
Context Specific

Pooled CRISPRi-mediated silencing of >1,000 transcriptional regulators expressed in single colorectal adenocarcinoma cells, followed by single-cell RNA-seq profiling at two time points, 1D and 4D, allowed reverse engineering the underlying tumor context-specific, causal regulatory network. Furthermore, the availability of experimentally derived, highly multiplexed gene reporter assays for each regulator, as identified by this analysis, allowed accurate assessment of differential protein activity following silencing of each regulator, thus providing proof-of-concept for generating comprehensive, tissue-specific networks of transcriptional and post-translational interactions. Analysis of this causal network allowed elucidation of complex autoregulatory mechanisms that have eluded previous computational approaches and supported systematic elucidation of cooperative mechanisms, where one regulatory protein can modulate the activity of another regulatory protein, as well as transcriptional mimicry, where one regulatory protein can phenocopy others.

scholarly journals RiboPlotR: a Visualization Tool for Periodic Ribo-seq Reads

2021 ◽  
Author(s):  
Hsin-Yen Larry Wu ◽  
Polly Yingshan Hsu
Keyword(s):  
Mrna Translation ◽  
Regulatory Mechanisms ◽  
Untranslated Regions ◽  
Data Display ◽  
Rna Seq ◽  
Genome Wide ◽  
Non Coding Rnas ◽  
Splice Junctions ◽  
Small Orfs ◽  
Reading Frames

Abstract Background: Ribo-seq has revolutionized the study of genome-wide mRNA translation. High-quality Ribo-seq data display strong 3-nucleotide (nt) periodicity, which corresponds to translating ribosomes deciphering three nts at a time. While 3-nt periodicity has been widely used to study novel translation events such as upstream ORFs in 5’ untranslated regions and small ORFs in presumed non-coding RNAs, tools that allow the visualization of these events remain underdeveloped.Results: RiboPlotR is a visualization package written in R that presents both RNA-seq coverage and Ribo-seq reads in genomic coordinates for all annotated transcript isoforms of a gene. Specifically, for individual isoform models, RiboPlotR plots Ribo-seq data related to splice junctions and presents the reads for all three reading frames in three different colors. Moreover, RiboPlotR shows Ribo-seq reads in upstream ORFs, 5' and 3' untranslated regions and introns, which is critical for observing new translation events and identifying potential regulatory mechanisms.Conclusions: RiboPlotR is freely available (https://github.com/hsinyenwu/RiboPlotR and https://sourceforge.net/projects/riboplotr/) and allows the visualization of translated features identified in Ribo-seq data.

scholarly journals Tracking pre-mRNA maturation across subcellular compartments identifies developmental gene regulation through intron retention and nuclear anchoring

2020 ◽  
Author(s):  
Kyu-Hyeon Yeom ◽  
Zhicheng Pan ◽  
Chia-Ho Lin ◽  
Hanyoung Lim ◽  
Wen Xiao ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Intron Retention ◽  
Developmental Regulation ◽  
Rna Seq ◽  
Protein Coding ◽  
Neuronal Progenitors ◽  
Subcellular Compartments ◽  
Cytoplasmic Rna ◽  
Mrna Maturation ◽  
Gene Transcripts

SUMMARYTo globally assess the distribution and processing of gene transcripts across subcellular compartments, we developed extensive RNA-seq datasets of both polyA+ and total RNA from chromatin, nucleoplasm and cytoplasm of mouse ESC, neuronal progenitors, and neurons. We identified protein-coding genes whose polyadenylated transcripts were more abundant in chromatin than cytoplasm. We defined introns exhibiting cotranscriptional splicing, complete intron retention in cytoplasmic RNA, and many introns retained in nucleoplasmic and chromatin RNA but absent from cytoplasmic RNA, including new introns controlled during neuronal development. In particular, we found that polyadenylated Gabbr1 transcripts are expressed in mESC but remain sequestered on chromatin until neuronal differentiation when they are processed and released to the cytoplasm. This developmental regulation of splicing and chromatin association demonstrates that the abundance of polyadenylated RNA is not always an indicator of functional gene expression. Our datasets provide a rich resource for analyzing many other aspects of mRNA maturation.

scholarly journals Regulation of DNA Methylation on Key Parasitism Genes of Cysticercus Cellulosae Revealed by Integrative Epigenomic-Transcriptomic Analyses

2020 ◽  
Author(s):  
Xinrui Wang ◽  
Weiyi Song ◽  
Yining Song ◽  
Guanyu Ji ◽  
Xuenong Luo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Developmental Regulation ◽  
Taenia Solium ◽  
Rna Seq ◽  
Host Interactions ◽  
Genome Wide ◽  
Cysticercus Cellulosae ◽  
Genes Encoding ◽  
Genome Bisulfite Sequencing ◽  
Parasitism Genes

Abstract Background: The life cycle of Taenia solium is characterized by different stages of development, requiring various kinds of hosts that can appropriately harbor the eggs (proglottids), the oncospheres, the larvae and the adults. Similar to other metazoan pathogens, T. solium undergoes transcriptional and developmental regulation via epigenetics during its complex lifecycle and host interactions.Result: In the present study, we integrated whole-genome bisulfite sequencing and RNA-seq technologies to characterize the genome-wide DNA methylation and its effect on transcription of Cysticercus cellulosae of T. solium. We confirm that the T. solium genome in the cysticercus stage is epigenetically modified by DNA methylation in a pattern similar to that of other invertebrate genomes, i.e., sparsely or moderately methylated. We also observed an enrichment of non-CpG methylation in defined genetic elements of the T. solium genome. Furthermore, an integrative analysis of both the transcriptome and the DNA methylome indicated a strong correlation between these two datasets, suggesting that gene expression might be tightly regulated by DNA methylation. Importantly, our data suggested that DNA methylation might play an important role in repressing key parasitism-related genes, including genes encoding excretion-secretion proteins, thereby raising the possibility of targeting DNA methylation processes as a useful strategy in therapeutics of cysticercosis.

scholarly journals Genome-wide Identification of Zero Nucleotide Recursive Splicing inDrosophila

2014 ◽  
Author(s):  
Michael O Duff ◽  
Sara Olson ◽  
Xintao Wei ◽  
Ahmad Osman ◽  
Alex Plocik ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Developmental Time ◽  
Rna Seq ◽  
Splice Sites ◽  
Sequencing Data ◽  
Genome Wide ◽  
Evolutionarily Conserved ◽  
And Function ◽  
Splice Junctions ◽  
Recursive Splicing

Recursive splicing is a process in which large introns are removed in multiple steps by resplicing at ratchet points - 5? splice sites recreated after splicing. Recursive splicing was first identified in the Drosophila Ultrabithorax (Ubx) gene and only three additional Drosophila genes have since been experimentally shown to undergo recursive splicing. Here, we identify 196 zero nucleotide exon ratchet points in 130 introns of 115 Drosophila genes from total RNA sequencing data generated from developmental time points, dissected tissues, and cultured cells. Recursive splicing events were identified by splice junctions that map to annotated 5? splice sites and unannotated intronic 3? splice sites, the presence of the sequence AG/GT at the 3? splice site, and a 5? to 3? gradient of decreasing RNA-Seq read density indicative of co-transcriptional splicing. The sequential nature of recursive splicing was confirmed by identification of lariat introns generated by splicing to and from the ratchet points. We also show that recursive splicing is a constitutive process, and that the sequence and function of ratchet points are evolutionarily conserved. Together these results indicate that recursive splicing is commonly used in Drosophila and provides insight into the mechanisms by which some introns are removed.

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