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

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.

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Extensive splicing across the Saccharomyces cerevisiae genome

10.1101/515163 ◽

2019 ◽

Cited By ~ 2

Author(s):

Stephen M. Douglass ◽

Calvin S. Leung ◽

Tracy L. Johnson

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Alternative Splice ◽

Rna Splicing ◽

Model Organism ◽

Growth Conditions ◽

Mrna Splicing ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Splice Forms

AbstractPre-mRNA splicing is vital for the proper function and regulation of eukaryotic gene expression. Saccharomyces cerevisiae has been used as a model organism for studies of RNA splicing because of the striking conservation of the spliceosome components and its catalytic activity. Nonetheless, there are relatively few annotated alternative splice forms, particularly when compared to higher eukaryotes. Here, we describe a method to combine large scale RNA sequencing data to accurately discover novel splice isoforms in Saccharomyces cerevisiae. Using our method, we find extensive evidence for novel splicing of annotated intron-containing genes as well as genes without previously annotated introns and splicing of transcripts that are antisense to annotated genes. By incorporating several mutant strains at varied temperatures, we find conditions which lead to differences in alternative splice form usage. Despite this, every class and category of alternative splicing we find in our datasets is found, often at lower frequency, in wildtype cells under normal growth conditions. Together, these findings show that there is widespread splicing in Saccharomyces cerevisiae, thus expanding our view of the regulatory potential of RNA splicing in yeast.Author SummaryPre-mRNA splicing is a fundamental step in eukaryotic gene expression. Saccharomyces cerevisiae, also known as brewer’s yeast, is a model organism for the study of pre-mRNA splicing in eukaryotes. Through the process of pre-mRNA splicing, a single gene is capable of encoding multiple mature mRNA products, but it is often difficult to identify the splice events that lead to these mRNA products. Here, we describe a method to accurately discover novel splice events in Saccharomyces cerevisiae and find evidence for extensive splicing in Saccharomyces. By utilizing a variety of strains and growth conditions, we are able to characterize many splice forms and correlate cellular conditions with prevalence of novel splice events.

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Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

Nature Communications ◽

10.1038/s41467-021-21150-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhe Sun ◽

Alexander V. Yakhnin ◽

Peter C. FitzGerald ◽

Carl E. Mclntosh ◽

Mikhail Kashlev

Keyword(s):

Environmental Cues ◽

Start Site ◽

Transcription Start ◽

Genome Wide Analysis ◽

Genome Wide ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Vitro Analysis ◽

In Vitro Analysis

AbstractPromoter-proximal pausing regulates eukaryotic gene expression and serves as checkpoints to assemble elongation/splicing machinery. Little is known how broadly this type of pausing regulates transcription in bacteria. We apply nascent elongating transcript sequencing combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Retention of σ70 induces strong backtracked pauses at a 10−20-bp distance from many promoters. The pauses in the 10−15-bp register of the promoter are dictated by the canonical −10 element, 6−7 nt spacer and “YR+1Y” motif centered at the transcription start site. The promoters for the pauses in the 16−20-bp register contain an additional −10-like sequence recognized by σ70. Our in vitro analysis reveals that DNA scrunching is involved in these pauses relieved by Gre cleavage factors. The genes coding for transcription factors are enriched in these pauses, suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

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SPLICE-q: a Python tool for genome-wide quantification of splicing efficiency

BMC Bioinformatics ◽

10.1186/s12859-021-04282-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Verônica R. de Melo Costa ◽

Julianus Pfeuffer ◽

Annita Louloupi ◽

Ulf A. V. Ørom ◽

Rosario M. Piro

Keyword(s):

Gene Expression ◽

Cancer Progression ◽

Time Course ◽

Progressive Increase ◽

Rna Seq ◽

Transcript Processing ◽

Aberrant Transcript ◽

Genome Wide ◽

Splicing Efficiency ◽

Nascent Rna

Abstract Background Introns are generally removed from primary transcripts to form mature RNA molecules in a post-transcriptional process called splicing. An efficient splicing of primary transcripts is an essential step in gene expression and its misregulation is related to numerous human diseases. Thus, to better understand the dynamics of this process and the perturbations that might be caused by aberrant transcript processing it is important to quantify splicing efficiency. Results Here, we introduce SPLICE-q, a fast and user-friendly Python tool for genome-wide SPLICing Efficiency quantification. It supports studies focusing on the implications of splicing efficiency in transcript processing dynamics. SPLICE-q uses aligned reads from strand-specific RNA-seq to quantify splicing efficiency for each intron individually and allows the user to select different levels of restrictiveness concerning the introns’ overlap with other genomic elements such as exons of other genes. We applied SPLICE-q to globally assess the dynamics of intron excision in yeast and human nascent RNA-seq. We also show its application using total RNA-seq from a patient-matched prostate cancer sample. Conclusions Our analyses illustrate that SPLICE-q is suitable to detect a progressive increase of splicing efficiency throughout a time course of nascent RNA-seq and it might be useful when it comes to understanding cancer progression beyond mere gene expression levels. SPLICE-q is available at: https://github.com/vrmelo/SPLICE-q

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Characterization of the radiation desiccation response regulon of the radioresistant bacterium Deinococcus radiodurans by integrative genomic analyses.

10.1101/2021.07.07.451423 ◽

2021 ◽

Author(s):

Nicolas Eugenie ◽

Yvan Zivanovic ◽

Gaelle Lelandais ◽

Genevieve Coste ◽

Claire Bouthier de la Tour ◽

...

Keyword(s):

Deinococcus Radiodurans ◽

Normal Growth ◽

Growth Conditions ◽

Rna Seq ◽

Genome Wide ◽

A Genome ◽

Genomic Analyses ◽

Number Of Genes ◽

Wide Scale

Numerous genes are overexpressed in the radioresistant bacterium Deinococcus radiodurans after exposure to radiation or prolonged desiccation. The DdrO and IrrE proteins play a major role in regulating the expression of approximately predicted twenty of these genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. Bioinformatic analyzes showed that this mechanism seems to be conserved in several species of Deinococcus, but many questions remain as such the number of genes regulated by DdrO. Here, by RNA-seq and CHiP-seq assays performed at a genome-wide scale coupled with bioinformatic analyses, we show that, the DdrO regulon in D. radiodurans includes many other genes than those previously described. These results thus pave the way to better understand the radioresistance mechanisms encoded by this bacterium.

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The Closely Related RNA helicases, UAP56 and URH49, Preferentially Form Distinct mRNA Export Machineries and Coordinately Regulate Mitotic Progression

Molecular Biology of the Cell ◽

10.1091/mbc.e09-10-0913 ◽

2010 ◽

Vol 21 (16) ◽

pp. 2953-2965 ◽

Cited By ~ 67

Author(s):

Tomohiro Yamazaki ◽

Naoko Fujiwara ◽

Hiroko Yukinaga ◽

Miki Ebisuya ◽

Takuya Shiki ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Mrna Export ◽

Mitotic Progression ◽

Rna Helicases ◽

Target Mrnas ◽

Genome Wide ◽

Chromatid Cohesion ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene

Nuclear export of mRNA is an essential process for eukaryotic gene expression. The TREX complex couples gene expression from transcription and splicing to mRNA export. Sub2, a core component of the TREX complex in yeast, has diversified in humans to two closely related RNA helicases, UAP56 and URH49. Here, we show that URH49 forms a novel URH49–CIP29 complex, termed the AREX (alternative mRNA export) complex, whereas UAP56 forms the human TREX complex. The mRNAs regulated by these helicases are different at the genome-wide level. The two sets of target mRNAs contain distinct subsets of key mitotic regulators. Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis. In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively. Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.

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Transposable element landscape in Drosophila populations selected for longevity

10.1101/867838 ◽

2019 ◽

Author(s):

Daniel K. Fabian ◽

Handan Melike Dönertaş ◽

Matías Fuentealba ◽

Linda Partridge ◽

Janet M. Thornton

Keyword(s):

Drosophila Melanogaster ◽

Telomere Maintenance ◽

Rna Seq ◽

Limited Evidence ◽

Negative Effects ◽

Genome Wide Analysis ◽

Genome Wide ◽

Age Dependent ◽

Genomic Insertions

ABSTRACTTransposable elements (TEs) inflict numerous negative effects on health and fitness as they replicate by integrating into new regions of the host genome. Even though organisms employ powerful mechanisms to demobilize TEs, transposons gradually lose repression during aging. The rising TE activity causes genomic instability and was implicated in age-dependent neurodegenerative diseases, inflammation and the determination of lifespan. It is therefore conceivable that long-lived individuals have improved TE silencing mechanisms resulting in reduced TE expression relative to their shorter-lived counterparts and fewer genomic insertions. Here, we test this hypothesis by performing the first genome-wide analysis of TE insertions and expression in populations of Drosophila melanogaster selected for longevity through late-life reproduction for 50-170 generations from four independent studies. Contrary to our expectation, TE families were generally more abundant in long-lived populations compared to non-selected controls. Although simulations showed that this was not expected under neutrality, we found little evidence for selection driving TE abundance differences. Additional RNA-seq analysis revealed a tendency for reducing TE expression in selected populations, which might be more important for lifespan than regulating genomic insertions. We further find limited evidence of parallel selection on genes related to TE regulation and transposition. However, telomeric TEs were genomically and transcriptionally more abundant in long-lived flies, suggesting improved telomere maintenance as a promising TE-mediated mechanism for prolonging lifespan. Our results provide a novel viewpoint indicating that reproduction at old age increases the opportunity of TEs to be passed on to the next generation with little impact on longevity.

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RiboPlotR: a Visualization Tool for Periodic Ribo-seq Reads

10.21203/rs.3.rs-944664/v1 ◽

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.

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Transposable Element Landscape in Drosophila Populations Selected for Longevity

Genome Biology and Evolution ◽

10.1093/gbe/evab031 ◽

2021 ◽

Vol 13 (4) ◽

Author(s):

Daniel K Fabian ◽

Handan Melike Dönertaş ◽

Matías Fuentealba ◽

Linda Partridge ◽

Janet M Thornton

Keyword(s):

Drosophila Melanogaster ◽

Telomere Maintenance ◽

Rna Seq ◽

Limited Evidence ◽

Negative Effects ◽

Genome Wide Analysis ◽

Genome Wide ◽

Age Dependent ◽

Genomic Insertions

Abstract Transposable elements (TEs) inflict numerous negative effects on health and fitness as they replicate by integrating into new regions of the host genome. Even though organisms employ powerful mechanisms to demobilize TEs, transposons gradually lose repression during aging. The rising TE activity causes genomic instability and was implicated in age-dependent neurodegenerative diseases, inflammation, and the determination of lifespan. It is therefore conceivable that long-lived individuals have improved TE silencing mechanisms resulting in reduced TE expression relative to their shorter-lived counterparts and fewer genomic insertions. Here, we test this hypothesis by performing the first genome-wide analysis of TE insertions and expression in populations of Drosophila melanogaster selected for longevity through late-life reproduction for 50–170 generations from four independent studies. Contrary to our expectation, TE families were generally more abundant in long-lived populations compared with nonselected controls. Although simulations showed that this was not expected under neutrality, we found little evidence for selection driving TE abundance differences. Additional RNA-seq analysis revealed a tendency for reducing TE expression in selected populations, which might be more important for lifespan than regulating genomic insertions. We further find limited evidence of parallel selection on genes related to TE regulation and transposition. However, telomeric TEs were genomically and transcriptionally more abundant in long-lived flies, suggesting improved telomere maintenance as a promising TE-mediated mechanism for prolonging lifespan. Our results provide a novel viewpoint indicating that reproduction at old age increases the opportunity of TEs to be passed on to the next generation with little impact on longevity.

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Natural antisense transcripts of MIR398 genes suppress microR398 processing and attenuate plant thermotolerance

Nature Communications ◽

10.1038/s41467-020-19186-x ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Yajie Li ◽

Xiaorong Li ◽

Jun Yang ◽

Yuke He

Keyword(s):

Genetic Manipulation ◽

Biological Processes ◽

Sequencing Data ◽

Antisense Transcripts ◽

Natural Antisense Transcripts ◽

Genome Wide ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Regulatory Loop ◽

Antisense Genes

Abstract MicroRNAs (miRNAs) and natural antisense transcripts (NATs) control many biological processes and have been broadly applied for genetic manipulation of eukaryotic gene expression. Still unclear, however, are whether and how NATs regulate miRNA production. Here, we report that the cis-NATs of MIR398 genes repress the processing of their pri-miRNAs. Through genome-wide analysis of RNA sequencing data, we identify cis-NATs of MIRNA genes in Arabidopsis and Brassica. In Arabidopsis, MIR398b and MIR398c are coexpressed in vascular tissues with their antisense genes NAT398b and NAT398c, respectively. Knock down of NAT398b and NAT398c promotes miR398 processing, resulting in stronger plant thermotolerance owing to silencing of miR398-targeted genes; in contrast, their overexpression activates NAT398b and NAT398c, causing poorer thermotolerance due to the upregulation of miR398-targeted genes. Unexpectedly, overexpression of MIR398b and MIR398c activates NAT398b and NAT398c. Taken together, these results suggest that NAT398b/c repress miR398 biogenesis and attenuate plant thermotolerance via a regulatory loop.

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Genome-wide Identification of Zero Nucleotide Recursive Splicing inDrosophila

10.1101/006163 ◽

2014 ◽

Cited By ~ 1

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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