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Quality control of spliced mRNAs requires the shuttling SR-proteins Gbp2 and Hrb1

ABSTRACT: Eukaryotic cells have to prevent the export of unspliced pre-mRNAs until intron removal is completed to avoid the expression of aberrant and potentially harmful proteins. Only mature RNAs associate with the export receptor Mex67 (mammalian TAP) and enter the cytoplasm. The underlying nuclear quality control mechanisms are still unclear. Here we show that two shuttling SR-proteins Gbp2 and Hrb1 are key surveillance factors for the selective export of spliced mRNAs in yeast. Their absence leads to the significant leakage of unspliced pre-mRNAs into the cytoplasm. They bind to pre-mRNAs and the spliceosome during splicing, where they are necessary for the surveillance of splicing and the stable binding of the TRAMP-complex to the spliceosome-bound transcripts. Faulty transcripts are marked for their degradation at the nuclear exosome. On correct mRNAs the SR-proteins recruit Mex67 upon completion of splicing to allow a quality controlled nuclear export. Altogether, these data identify a role for shuttling SR-proteins in mRNA surveillance and nuclear mRNA quality control.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE52808 | GEO | 2013/12/04

SECONDARY ACCESSION(S): PRJNA230517

REPOSITORIES: GEO

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Quality control of spliced mRNAs requires the shuttling SR-proteins Gbp2 and Hrb1

Project description:Eukaryotic cells have to prevent the export of unspliced pre-mRNAs until intron removal is completed to avoid the expression of aberrant and potentially harmful proteins. Only mature RNAs associate with the export receptor Mex67 (mammalian TAP) and enter the cytoplasm. The underlying nuclear quality control mechanisms are still unclear. Here we show that two shuttling SR-proteins Gbp2 and Hrb1 are key surveillance factors for the selective export of spliced mRNAs in yeast. Their absence leads to the significant leakage of unspliced pre-mRNAs into the cytoplasm. They bind to pre-mRNAs and the spliceosome during splicing, where they are necessary for the surveillance of splicing and the stable binding of the TRAMP-complex to the spliceosome-bound transcripts. Faulty transcripts are marked for their degradation at the nuclear exosome. On correct mRNAs the SR-proteins recruit Mex67 upon completion of splicing to allow a quality controlled nuclear export. Altogether, these data identify a role for shuttling SR-proteins in mRNA surveillance and nuclear mRNA quality control. 6 samples, i.e. 2 replicates per protein Gbp2, Hrb1 and Npl3

2013-12-04 | E-GEOD-52808 | biostudies-arrayexpress

The shuttling SR-like proteins Gbp2 and Hrb1 mediate the nuclear export of spliced mRNAs

Project description:Three shuttling SR-like proteins exist in Saccharomyces cerevisiae, Npl3, Gbp2 and Hrb1, that are involved in the nuclear export of mRNAs. In a screen for genes that regulate the export of Gbp2, we identified novel mutants of the splicing factors PRP8 and PRP17 that lead to severe mislocalization defects for Gbp2 and Hrb1, but not Npl3. Microarray and qRT-PCR analyses show that Gbp2 and Hrb1 preferentially bind to transcripts derived from intron-containing genes. Moreover, in contrast to Npl3, Gbp2 and Hrb1 show genetic and physical interactions with late splicing factors such as Prp17 and Prp43. Further, RNA co-immunoprecipitation experiments reveal that, unlike Npl3, association of Gbp2 and Hrb1 with the mRNA requires splicing, and this in turn is required for their Mex67 recruitment. We propose a model in which Gbp2 and Hrb1 are attached to the mRNAs at late stages of splicing to promote the subsequent export of spliced mRNAs. keyword: RIP-chip RNA-IP of endogenously expressed 3-fold C-terminal myc-tagged Gbp2 and Hrb1 in S288C background cells was each performed once in cells grown to a density of 4x10^7 cells/ml and hybridized against a total (input) RNA reference.

2013-08-30 | E-GEOD-20738 | biostudies-arrayexpress

Trypanosomes can initiate nuclear export co-transcriptionally

Project description:The nuclear envelope serves as important mRNA surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualised nuclear export in trypanosomes by probing large, endogenous mRNA by intramolecular multi-colour single molecule FISH (smFISH). In addition, unspliced mRNAs were visualised by co-probing two adjacent introns or intergenic regions. We found that the initation of nuclear export requires neither the completion of transcription nor trans-splicing. Nevertheless, the inhibition of trans-splicing blocked cytoplasmic transport of the of unspliced mRNAs and only a small fraction reached the nucleus-distant cytoplasm. Most of the unspliced transcripts remained at the nuclear periphery, within transport and in nuclear periphery granules (NPGs) localised at the cytoplasmic site of nuclear pores that resemble stress granules in composition. Our work shows that, in striking contrast to other eukaryotes, trypanosomes can start nuclear export while the mRNA is still transcribed, but unspliced transcripts remain ‘stuck’ in nuclear pores, probably awaiting processing or decay. Our data indicate that trypanosomes regulate the completion of nuclear export rather than the start.

2018-10-26 | PXD010143 | Pride

The Deubiquitylase Ubp15 Couples Transcription to mRNA Export

Project description:The nuclear export of messenger RNAs (mRNAs) is intimately coupled to their synthesis. pre-mRNAs assemble into dynamic ribonucleoparticles as they are being transcribed, processed and exported. The role of ubiquitylation in this process is increasingly recognized as the ubiquitylation of many key players have been shown to affect mRNA nuclear export. While a few E3 ligases have been shown to regulate nuclear export, evidence for deubiquitylases is currently lacking. Here, we identified the deubiquitylase Ubp15 as a regulator of nuclear export in Saccharomyces cerevisiae. Ubp15 interacts both with RNA polymerase II and with the nuclear pore complex, and its deletion reverts the nuclear export defect of mutants of the E3 ligase Rsp5. The deletion of UBP15 leads to hyper-ubiquitylation of the main nuclear export receptor Mex67 and affects its association with THO, a complex coupling transcription to mRNA processing and involved in the recruitment of mRNA export factors to nascent transcripts. Collectively, our data support a role for Ubp15 in coupling transcription to mRNA export.

2020-11-23 | GSE154671 | GEO

Transcriptome-wide mapping of RNA:protein interactions of seven SR proteins and NXF1 in P19 cells by iCLIP

Project description:We used GFP-tagged SR proteins expressed at endogenous levels and iCLIP to identify and compare endogenous RNA targets of individual SR proteins, map the preferential sites of binding, compare binding pattern and binding motifs between family members and to NXF1 and quantify binding of SR proteins and NXF1 to spliced versus unspliced RNAs to study the role of SR proteins in mRNA export via NXF1.

2016-03-07 | GSE69689 | GEO

Double stranded RNA formation leads to preferential nuclear export and gene expression

Project description:mRNAs are transcribed and processed in the nucleus before they are exported into the cytoplasm for translation. Export is mediated by the export receptor heterodimer Mex67-Mtr2 in yeast (TAP-p15 in humans). Interestingly, also many lncRNAs leave the nucleus but it is currently unclear why they travel into the cytoplasm. Here we show that antisense (as)RNAs accelerate mRNA export by annealing with their sense counterparts through the helicase Dbp2. These double-stranded (ds)RNAs dominate export compared to single-stranded (ss)RNA, as they have a higher capacity and affinity for the export receptor Mex67. In this way, asRNAs boost gene expression, which is beneficial for cells. This is particularly important upon expression program changes. Consequently, the degradation or prevention of the formation of dsRNA is toxic for cells. This mechanism illuminates the general cellular occurrence of asRNAs and explains their nuclear export.

2024-04-30 | GSE252951 | GEO

TPR Knockdown Frac-Seq

Project description:How splicing regulates the nuclear export of mRNAs has been a source of much debate. While splicing has been shown to enhance nuclear export, it has remained unclear whether mRNAs generated from intronless genes use specific machinery to promote their export. Here we investigate the role of the major nuclear pore basket protein, TPR, in regulating mRNA and lncRNA nuclear export. We provide evidence that TPR is required for the nuclear export of mRNAs and lncRNAs that are generated from intronless or intron-poor genes. In contrast, TPR is not required for the nuclear retention of mRNAs that have retained introns, or unused 5’ splice site motifs. In summary, our study provides one of the first examples of a factor that is specifically required for the nuclear export of intronless mRNAs.

2020-10-02 | GSE135537 | GEO

Saccharomyces cerevisiae

Project description:Quality control of spliced mRNAs requires the shuttling SR-proteins Gbp2 and Hrb1

| PRJNA230517 | ENA

RIP-chip by array of mouse undifferentiated and neural cells to identify mRNAs associated with SRp20 and SRp75

Project description:Members of the SR protein family of RNA binding proteins play numerous roles in gene expression, including the regulation of pre-mRNA splicing, mRNA export, and translation. How SR proteins coordinate gene expression programs is unknown, and comprehensive knowledge of endogenous mRNA targets is lacking. We established physiological expression of GFP-tagged SR protein, allowing the immunopurification and systematic identification of mRNAs associated with SRp20 and SRp75. This genome-wide analysis showed that SRp20 and SRp75 are components of distinct, mostly non-overlapping mRNPs in undifferentiated and neural cells.

2010-07-20 | E-MEXP-2637 | biostudies-arrayexpress

Control of gene expression by Ott1/RBM15 protein

Project description:The conserved mRNA nuclear export receptor NXF1 (Mex67 in yeast) assembles with messenger ribonucleoproteins (mRNP) in the nucleus and guides them through the nuclear pore complex (NPC) into the cytoplasm. The DEAD-box RNA helicase Dbp5 is essential for nuclear export of mRNA and is thought to dissociate Mex67 from mRNP upon translocation, thereby generating directional passage. However, the molecular mechanism by which Dbp5 recognizes Mex67-containing mRNP is not clear. Here we report that the human NXF1-binding protein RBM15 binds to DBP5 specifically and enables its direct contact with mRNA. We found that RBM15 is enriched at the nuclear envelope and colocalizes with DBP5 at the NPC. Knockout of RBM15’s orthologue in mouse (Ott1) leads to global changes in mRNA expression and excessive mRNA accumulation in the cytoplasm, indicating an essential role of RBM15 in mRNA export regulation. We propose that RBM15 acts locally at the NPC, by transiently bridging the NXF1-mRNP complexes to DBP5, thereby contributing to the substrate specificity of mRNA export.

2008-06-18 | GSE11785 | GEO

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