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Nonsense Mediated mRNA decay mutes the splicing defects of spliceosome component mutations

ABSTRACT: The role of many splicing factors in pre-mRNA splicing and the involvement of these factors in the processing of specific transcripts have often been defined through the analysis of loss of function mutants in vivo. Here we show that inactivating the nonsense mediated mRNA decay (NMD) results in an enhancement of splicing phenotypes associated with several splicing factors mutations. Tiling microarrays showed that inactivation of the NMD factor Upf1p in the prp17Î and prp18Î mutant strains reveals a larger spectrum of splicing defects than what is observed in the single mutants, including new transcripts previously shown unaffected by Prp17p or Prp18p inactivation. In addition, inactivation of Upf1 in the prp22-1 mutant enhances the unspliced precursor accumulation phenotype of several specific transcripts and partially suppresses growth defects associated with the prp17Î or prp22-1 mutations. These results support the idea that RNA surveillance by NMD mutes some of the effects of splicing factors mutations and show that the roles of splicing factors and their transcripts specificity cannot be fully understood in vivo unless RNA degradation systems that degrade unspliced precursors are also inactivated. Three samples from the Prp17delta, Prp18delta, Prp17deltaUpf1delta and Prp18Upf1delta mutants were grown indepedently and analyzed by tiling arrays to understand the role of the NMD component Upf1 in minimizing the accumulation of unspliced RNAs generated by the Prp17delta and Prp18delta splicing mutations.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Matteo Pellegrini

PROVIDER: E-GEOD-18288 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Nonsense Mediated mRNA decay mutes the splicing defects of spliceosome component mutations

Project description:The role of many splicing factors in pre-mRNA splicing and the involvement of these factors in the processing of specific transcripts have often been defined through the analysis of loss of function mutants in vivo. Here we show that inactivating the nonsense mediated mRNA decay (NMD) results in an enhancement of splicing phenotypes associated with several splicing factors mutations. Tiling microarrays showed that inactivation of the NMD factor Upf1p in the prp17Δ and prp18Δ mutant strains reveals a larger spectrum of splicing defects than what is observed in the single mutants, including new transcripts previously shown unaffected by Prp17p or Prp18p inactivation. In addition, inactivation of Upf1 in the prp22-1 mutant enhances the unspliced precursor accumulation phenotype of several specific transcripts and partially suppresses growth defects associated with the prp17Δ or prp22-1 mutations. These results support the idea that RNA surveillance by NMD mutes some of the effects of splicing factors mutations and show that the roles of splicing factors and their transcripts specificity cannot be fully understood in vivo unless RNA degradation systems that degrade unspliced precursors are also inactivated.

2009-12-31 | GSE18288 | GEO

Nonsense-mediated decay of alternative precursor mRNA splicing variants is a major determinant of the eukaryotic steady state transcriptome

Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes aberrant transcripts arising from mutations and transcriptional mistakes. Moreover, differential transcript processing such as alternative precursor mRNA splicing (AS) can generate NMD substrates, however, the extent of coupled AS and NMD remained unclear. To investigate NMD targeting of AS variants, we performed transcriptome-wide splicing studies using Arabidopsis thaliana single and double mutants in the NMD factor homologues UPF1 and UPF3, as well as samples treated with the translation inhibitor cycloheximide (CHX). Our analyses revealed that at least 17.5% of all multi-exon, protein-coding genes produce splicing variants of all major types that are targeted by NMD, with a significant fraction originating from the splicing of cryptic introns. Furthermore, accumulation of many intron-retained mRNAs in the mutants, but not in response to CHX suggests the action of distinct routes of NMD with variable impact of translation. Importantly, 92.3% of the NMD-responsive mRNAs exhibit classical NMD-eliciting features, supporting their authenticity as direct targets. NMD-dependent AS variants are linked to diverse biological functions, including the poison exon-mediated regulation of signaling and posttranslational protein modification components. In addition to mRNAs, numerous non-coding RNAs as well as newly identified transcripts derived from intergenic regions were shown to be NMD responsive. In summary, our comprehensive analysis of AS-coupled NMD provides strong evidence for a major function of this pathway in shaping the transcriptome, having fundamental implications in gene regulation and quality control of transcript processing steps in higher eukaryotes. Comparison of gene expression and alternative splicing patterns in control and nonsense-mediated decay (NMD)-impaired Arabidopsis seedlings

2013-10-01 | E-GEOD-41432 | biostudies-arrayexpress

Pervasive changes of mRNA splicing in upf1 deficient zebrafish identify rpl10a as a regulator of T cell development

Project description:The transcriptome of eukaryotic cells is constantly monitored for errors to avoid the production of undesired protein variants. The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway degrades aberrant mRNAs, but also functions in the regulation of transcript abundance in response to changed physiological states. As a result of its essential functions, only few animal models of impaired NMD activity have been developed. Here, we describe a zebrafish mutant of upf1, encoding the central component of the NMD machinery. Fish homozygous for the upf1t20450 allele (Y163X) survive until day 10 after fertilization, presenting with impaired T cell development as one of the most conspicuous features of the mutant phenotype. Analysis of differentially expressed genes identified dysregulation of the pre-mRNA splicing pathway, accompanied by perturbed auto-regulation of canonical splicing activators (SRSF) and repressors (HNRNP). In upf1-deficient mutants, NMD-susceptible transcripts of ribosomal proteins that are known for their role as non-canonical splicing regulators were greatly increased, most notably, rpl10a. When the levels of NMD-susceptible rpl10a transcripts were artifically increased in zebrafish morphants, T cell development was significantly impaired, suggesting that perturbed autoregulation of rpl10a splicing contributes to failing T cell development in upf1 deficiency. Our results identify an extra-ribosomal tissue-specific function to rpl10a in the immune system, and thus exemplify the advantages of the zebrafish model to study the effects of upf1-deficiency in the context of a vertebrate organism.

2020-07-13 | GSE136669 | GEO

Nonsense-mediated decay of alternative precursor mRNA splicing variants is a major determinant of the eukaryotic steady state transcriptome

2013-10-01 | GSE41432 | GEO

The origins and consequences of UPF1 variants in pancreatic adenosquamous carcinoma

Project description:Pancreatic adenosquamous carcinoma (PASC) is an aggressive cancer whose mutational origins are poorly understood. An early study reported high-frequency somatic mutations affecting UPF1, a core nonsense-mediated mRNA decay (NMD) factor, in PASC, but subsequent studies did not observe these lesions. The corresponding controversy about whether UPF1 mutations are important contributors to PASC has been exacerbated by a paucity of functional studies. Here, we modeled two UPF1 mutations to find no significant effects on pancreatic cancer growth, acquisition of adenosquamous features, UPF1 splicing, UPF1 protein levels, or NMD efficiency. We subsequently discovered that ~40% of UPF1 mutations reportedly present in PASCs are identical to standing genetic variants in the human population, suggesting that they may be non-pathogenic inherited variants rather than pathogenic mutations. Our data suggest that UPF1 is not a common functional driver of PASC and motivate further attempts to identify unique genetic features defining these malignancies.

2020-12-19 | GSE163517 | GEO

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency 2

Project description:The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited “closed” state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active “open” state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

2024-05-29 | GSE246484 | GEO

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency 1

2024-05-29 | GSE246483 | GEO

Effect of nonsense-mediated decay inhibition on expression of 3' UTR spliced transcripts in colorectal carcinoma cell line HCT116

Project description:The presence of an exon junction more than 55nt downstream of a stop codon is generally considered to be a trigger for nonsense-mediated decay (NMD). Thus, transcripts containing 3’UTR introns (3UIs) are often considered as transcriptional noise. Interestingly, upon knockdown of NMD factor UPF1 we see examples of both increased and decreased expression of the 3UI-spliced isoforms. In the oncogene HRAS, upon UPF1 knockdown, we see the 3UI-retaining isoform increases in expression where the spliced isoform decreases, suggesting that 3UI splicing protects the transcript against UPF1-mediated degradation.

2024-12-20 | GSE251666 | GEO

Genes co-regulated by the NMD factors UPF2 and SMG5

Project description:Eukaryotic cells have evolved a mechanism called nonsense mediated mRNA decay (NMD) that detects and degrades aberrant mRNAs that contain premature termination codons (PTCs). NMD has recently acquired broader importance as it has been found to regulate not only aberrant mRNAs but also a great diversity of transcripts, including wild type genes in mammals, Drosophila and yeast. Seven proteins are the core of the NMD complex: SMG1, SMG2 (UPF1), SMG3 (UPF2), SMG4 (UPF3), SMG5, SMG6 and SMG7. Plants have orthologues of most of these proteins. We have identified and characterized a number of alleles of UPF1, UPF3 and SMG5 and made 35S:UPF2 RNAi (upf2i) transgenic plants, all of which share some phenotypic characteristics. Transcriptome analysis of upf1 and upf3 mutants has identified a subset of genes coregulated by UPF1 and UPF3 and, therefore, by NMD (unpublished data). By doing further transcriptome analysis on smg5 mutants and upf2i plants we aim to build a more robust subset of NMD targets in Arabidopsis. RNA will be extracted from 17 day-old mutant, transgenic and wild type seedlings grown at 22-24 C under constant light. 6 samples were used in this experiment

2011-10-06 | E-GEOD-32671 | biostudies-arrayexpress

Widespread Use of Non-Productive Alternative Splice Sites in Saccharomyces cerevisiae

Project description:Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many Saccharomyces cerevisiae intron-containing genes exhibit usage of alternative splice sites, but most transcripts generated by splicing from these sites are non-functional because they introduce premature termination codons leading to transcript degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3M-bM-^@M-^Y splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3M-bM-^@M-^Y-splice site. The use of non-productive alternative splice sites can limit the expression of some transcripts and can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results reveal that alternative splicing is frequent in S.cerevisiae but masked by RNA degradation and that the use of alternative splice sites is mostly aimed at controlling transcript levels rather than increasing proteome diversity. mRNA-Seq profiling of 3 mutants in the nonsense-mediated mRNA decay pathway and wildtype yeast

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