scholarly journals A Collection of Pre-mRNA Splicing Mutants in Arabidopsis thaliana

2020 ◽  
Vol 10 (6) ◽  
pp. 1983-1996 ◽  
Author(s):  
Tatsuo Kanno ◽  
Peter Venhuizen ◽  
Ming-Tsung Wu ◽  
Phebe Chiou ◽  
Chia-Liang Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Alternative Splicing ◽  
Reporter Gene ◽  
Rna Binding ◽  
Binding Protein ◽  
Genetic Screen ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Sequencing Data

To investigate factors influencing pre-mRNA splicing in plants, we conducted a forward genetic screen using an alternatively-spliced GFP reporter gene in Arabidopsis thaliana. This effort generated a collection of sixteen mutants impaired in various splicing-related proteins, many of which had not been recovered in any prior genetic screen or implicated in splicing in plants. The factors are predicted to act at different steps of the spliceosomal cycle, snRNP biogenesis pathway, transcription, and mRNA transport. We have described eleven of the mutants in recent publications. Here we present the final five mutants, which are defective, respectively, in RNA-BINDING PROTEIN 45D (rbp45d), DIGEORGE SYNDROME CRITICAL REGION 14 (dgcr14), CYCLIN-DEPENDENT KINASE G2 (cdkg2), INTERACTS WITH SPT6 (iws1) and CAP BINDING PROTEIN 80 (cbp80). We provide RNA-sequencing data and analyses of differential gene expression and alternative splicing patterns for the cbp80 mutant and for several previously published mutants, including smfa and new alleles of cwc16a, for which such information was not yet available. Sequencing of small RNAs from the cbp80 mutant highlighted the necessity of wild-type CBP80 for processing of microRNA (miRNA) precursors into mature miRNAs. Redundancy tests of paralogs encoding several of the splicing factors revealed their functional non-equivalence in the GFP reporter gene system. We discuss the cumulative findings and their implications for the regulation of pre-mRNA splicing efficiency and alternative splicing in plants. The mutant collection provides a unique resource for further studies on a coherent set of splicing factors and their roles in gene expression, alternative splicing and plant development.

scholarly journals Alternative splicing landscape of the neural transcriptome in a cytoplasmic-predominant Pten expression murine model of autism-like Behavior

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Stetson Thacker ◽  
Marilyn Sefyi ◽  
Charis Eng
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Murine Model ◽  
Neurodevelopmental Disorders ◽  
Autism Spectrum ◽  
Splicing Factors ◽  
Sequencing Data ◽  
Central Nervous Systems ◽  
Functional Complexity ◽  
The Brain

Abstract Alternative splicing (AS) is a posttranscriptional mechanism regulating gene expression that complex organisms utilize to expand proteome diversity from a comparatively limited set of genes. Recent research has increasingly associated AS with increased functional complexity in the central nervous systems in higher order mammals. This work has heavily implicated aberrant AS in several neurocognitive and neurodevelopmental disorders, including autism. Due to the strong genetic association between germline PTEN mutations and autism spectrum disorder (ASD), we hypothesized that germline PTEN mutations would alter AS patterns, contributing to the pathophysiology of ASD. In a murine model of constitutional mislocalization of Pten, recapitulating an autism-like phenotype, we found significant changes in AS patterns across the neural transcriptome by analyzing RNA-sequencing data with the program rMATS. A few hundred significant alternative splicing events (ASEs) that differentiate each m3m4 genotype were identified. These ASEs occur in genes enriched in PTEN signaling, inositol metabolism, and several other pathways relevant to the pathophysiology of ASD. In addition, we identified expression changes in several splicing factors known to be enriched in the nervous system. For instance, the master regulator of microexons, Srrm4, has decreased expression, and consequently, we found decreased inclusion of microexons in the Ptenm3m4/m3m4 cortex (~10% decrease). We also demonstrated that the m3m4 mutation disrupts the interaction between Pten and U2af2, a member of the spliceosome. In sum, our observations point to germline Pten disruption changing the landscape of alternative splicing in the brain, and these changes may be relevant to the pathogenesis and/or maintenance of PTEN-ASD phenotypes.

scholarly journals Replication stress-induced alternative mRNA splicing alters properties of the histone RNA-binding protein HBP/SLBP: a key factor in the control of histone gene expression

2013 ◽  
Vol 33 (5) ◽  
Author(s):  
Alexander M. J. Rattray ◽  
Pamela Nicholson ◽  
Berndt Müller
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Replication Stress ◽  
S Phase ◽  
Histone Gene ◽  
Histone Genes ◽  
Histone Gene Expression

Animal replication-dependent histone genes produce histone proteins for the packaging of newly replicated genomic DNA. The expression of these histone genes occurs during S phase and is linked to DNA replication via S-phase checkpoints. The histone RNA-binding protein HBP/SLBP (hairpin-binding protein/stem-loop binding protein), an essential regulator of histone gene expression, binds to the conserved hairpin structure located in the 3′UTR (untranslated region) of histone mRNA and participates in histone pre-mRNA processing, translation and histone mRNA degradation. Here, we report the accumulation of alternatively spliced HBP/SLBP transcripts lacking exons 2 and/or 3 in HeLa cells exposed to replication stress. We also detected a shorter HBP/SLBP protein isoform under these conditions that can be accounted for by alternative splicing of HBP/SLBP mRNA. HBP/SLBP mRNA alternative splicing returned to low levels again upon removal of replication stress and was abrogated by caffeine, suggesting the involvement of checkpoint kinases. Analysis of HBP/SLBP cellular localization using GFP (green fluorescent protein) fusion proteins revealed that HBP/SLBP protein and isoforms lacking the domains encoded by exon 2 and exons 2 and 3 were found in the nucleus and cytoplasm, whereas HBP/SLBP lacking the domain encoded by exon 3 was predominantly localised to the nucleus. This isoform lacks the conserved region important for protein–protein interaction with the CTIF [CBP80/20 (cap-binding protein 80/20)]-dependent initiation translation factor and the eIF4E (eukaryotic initiation factor 4E)-dependent translation factor SLIP1/MIF4GD (SLBP-interacting protein 1/MIF4G domain). Consistent with this, we have previously demonstrated that this region is required for the function of HBP/SLBP in cap-dependent translation. In conclusion, alternative splicing allows the synthesis of HBP/SLBP isoforms with different properties that may be important for regulating HBP/SLBP functions during replication stress.

scholarly journals An RNA Switch of a Large Exon of Ninein Is Regulated by the Neural Stem Cell Specific-RNA Binding Protein, Qki5

2019 ◽  
Vol 20 (5) ◽  
pp. 1010 ◽  
Author(s):  
Yoshika Hayakawa-Yano ◽  
Masato Yano
Keyword(s):  
Alternative Splicing ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Neural Progenitor Cell ◽  
Conditional Knockout ◽  
Splicing Factors ◽  
Axon Development ◽  
Rna Switch

A set of tissue-specific splicing factors are thought to govern alternative splicing events during neural progenitor cell (NPC)-to-neuron transition by regulating neuron-specific exons. Here, we propose one such factor, RNA-binding protein Quaking 5 (Qki5), which is specifically expressed in the early embryonic neural stem cells. We performed mRNA-SEQ (Sequence) analysis using mRNAs obtained by developing cerebral cortices in Qk (Quaking) conditional knockout (cKO) mice. As expected, we found a large number of alternative splicing changes between control and conditional knockouts relative to changes in transcript levels. DAVID (The Database for Annotation, Visualization and Integrated Discovery) and Metascape analyses suggested that the affected spliced genes are involved in axon development and microtubule-based processes. Among these, the mRNA coding for the Ninein protein is listed as one of Qki protein-dependent alternative splicing targets. Interestingly, this exon encodes a very long polypeptide (2121 nt), and has been previously defined as a dynamic RNA switch during the NPC-to-neuron transition. Additionally, we validated that the regulation of this large exon is consistent with the Qki5-dependent alternative exon inclusion mode suggested by our previous Qki5 HITS-CLIP (high throughput sequencing-cross linking immunoprecipitation) analysis. Taken together, these data suggest that Qki5 is an important factor for alternative splicing in the NPC-to-neuron transition.

scholarly journals An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana

2012 ◽  
Vol 40 (22) ◽  
pp. 11240-11255 ◽  
Author(s):  
Corinna Streitner ◽  
Tino Köster ◽  
Craig G. Simpson ◽  
Paul Shaw ◽  
Selahattin Danisman ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein

scholarly journals Poly(A) Polymerase and the Nuclear Poly(A) Binding Protein, PABPN1, Coordinate the Splicing and Degradation of a Subset of Human Pre-mRNAs

2015 ◽  
Vol 35 (13) ◽  
pp. 2218-2230 ◽  
Author(s):  
Lisa Muniz ◽  
Lee Davidson ◽  
Steven West
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Mrna Splicing ◽  
Oculopharyngeal Muscular Dystrophy ◽  
Splicing Factors ◽  
Human Genes ◽  
Protein Encoding ◽  
Cleavage And Polyadenylation ◽  
Regulatory Potential ◽  
Terminal Poly

Most human protein-encoding transcripts contain multiple introns that are removed by splicing. Although splicing catalysis is frequently cotranscriptional, some introns are excised after polyadenylation. Accumulating evidence suggests that delayed splicing has regulatory potential, but the mechanisms are still not well understood. Here we identify a terminal poly(A) tail as being important for a subset of intron excision events that follow cleavage and polyadenylation. In these cases, splicing is promoted by the nuclear poly(A) binding protein, PABPN1, and poly(A) polymerase (PAP). PABPN1 promotes intron excision in the context of 3′-end polyadenylation but not when bound to internal A-tracts. Importantly, the ability of PABPN1 to promote splicing requires its RNA binding and, to a lesser extent, PAP-stimulatory functions. Interestingly, an N-terminal alanine expansion in PABPN1 that is thought to cause oculopharyngeal muscular dystrophy cannot completely rescue the effects of PABPN1 depletion, suggesting that this pathway may have relevance to disease. Finally, inefficient polyadenylation is associated with impaired recruitment of splicing factors to affected introns, which are consequently degraded by the exosome. Our studies uncover a new function for polyadenylation in controlling the expression of a subset of human genes via pre-mRNA splicing.

scholarly journals Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing

2021 ◽  
Author(s):  
Kevin Tsai ◽  
Hal P. Bogerd ◽  
Edward M. Kennedy ◽  
Ann Emery ◽  
Ronald Swanstrom ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Stability ◽  
Rna Binding Protein ◽  
Viral Gene ◽  
Viral Mrnas ◽  
Wide Range ◽  
Hiv 1

AbstractPrevious work in several laboratories has demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of DNA and RNA viruses, yet the underlying mechanisms responsible for this positive effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how m6A readers regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is in large part mediated by the the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs while YTHDF2 binding correlates with increased HIV-1 transcript stability.Author SummaryThis manuscript reports that the expression of mRNAs encoded by the pathogenic human retrovirus HIV-1 is regulated by the methylation of a small number of specific adenosine residues. These in turn recruit a nuclear RNA binding protein, called YTHDC1, which modulates the alternative splicing of HIV-1 transcripts, as well as a cytoplasmic RNA binding protein, called YTHDF2, which stabilizes viral mRNAs. The regulation of HIV-1 gene expression by adenosine methylation is therefore critical for the effective and ordered expression of HIV-1 mRNAs and could represent a novel target for antiviral development.

scholarly journals Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Guiomar Martín ◽  
Yamile Márquez ◽  
Federica Mantica ◽  
Paula Duque ◽  
Manuel Irimia
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Alternative Splicing ◽  
Stress Responses ◽  
Target Genes ◽  
Intron Retention ◽  
Single Gene ◽  
Exon Skipping ◽  
Environmental Response ◽  
Biotic Stresses

Abstract Background Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing. Results We generate a massive resource for Arabidopsis thaliana, PastDB, comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion. Conclusions Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in A. thaliana.

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