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MKRN2 physically interacts with GLE1 to regulate mRNA export and zebrafish retinal development (RNA-Seq)

ABSTRACT: The canonical mammalian mRNA export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through mRNP remodeling. We conducted a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network revealed unexpected interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assessed MKRN2 epistasis with GLE1 in a genetically tractable zebrafish model. Strikingly, morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescued retinal developmental defects seen upon GLE1 depletion. Next, using iCLIP, we showed that MKRN2 binds preferentially to the 3'UTR of a diverse subset of mRNAs. Next-generation sequencing of fractionated cell extracts revealed that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, these results indicate that MKRN2 selectively interacts with GLE1 to regulate mRNA nuclear export and retinal development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE136421 | GEO | 2020/05/19

REPOSITORIES: GEO

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MKRN2 physically interacts with GLE1 to regulate mRNA export and zebrafish retinal development

Project description:The canonical mammalian mRNA export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through mRNP remodeling. We conducted a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network revealed unexpected interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assessed MKRN2 epistasis with GLE1 in a genetically tractable zebrafish model. Strikingly, morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescued retinal developmental defects seen upon GLE1 depletion. Next, using iCLIP, we showed that MKRN2 binds preferentially to the 3'UTR of a diverse subset of mRNAs. Next-generation sequencing of fractionated cell extracts revealed that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, these results indicate that MKRN2 selectively interacts with GLE1 to regulate mRNA nuclear export and retinal development.

2020-05-19 | GSE136399 | GEO

Human RNA-binding protein E3 ubiquitin ligase AP-MS

Project description:The canonical mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conducted a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network revealed interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assessed MKRN2 epistasis with GLE1 in a genetically tractable zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescued retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we showed that MKRN2 binds selectively to the 3'UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.

2020-05-29 | PXD017541 | 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

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

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

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

Project description:The nuclear phase of the gene expression pathway culminates in the export of mature mRNAs to the cytoplasm through nuclear pore complexes (NPCs). GANP (Germinal-centre Associated Nuclear Protein) promotes the transfer to NPCs of mRNAs bound to the transport factor NXF1. Here, we demonstrate that GANP, subunit of the TREX-2 mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. We used gene expression profiling to compare the abundance of cytoplasmic RNAs after GANP or NXF1 depletion

2014-02-14 | E-GEOD-54481 | biostudies-arrayexpress

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

Nuclear export is a limiting factor in eukaryotic mRNA metabolism

Project description:The eukaryotic mRNA life cycle includes transcription, nuclear mRNA export and degradation. To quantify all these processes simultaneously, we perform thiol-linked alkylation after metabolic labeling of RNA with 4-thiouridine (4sU), followed by sequencing of RNA (SLAM-seq) in the nuclear and cytosolic compartments. We develop a model that reliably quantifies mRNA synthesis, nuclear export, and nuclear and cytosolic degradation rates on a genome-wide scale. We find that nuclear degradation of polyadenylated mRNA is negligible and nuclear mRNA export is slow, while cytosolic mRNA degradation is comparatively fast. Consequently, an mRNA molecule generally spends most of its life in the nucleus. We also observe large differences in the nuclear export rates of different 3’UTR transcript isoforms. Furthermore, we identify genes whose expression is abruptly induced upon metabolic labeling. These transcripts are exported substantially faster than average mRNAs, suggesting the existence of alternative export pathways. Our results highlight nuclear mRNA export as a limiting factor in mRNA metabolism and gene regulation.

2024-04-23 | GSE233546 | GEO

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

2014-02-14 | GSE54481 | GEO

MKRN2 physically interacts with GLE1 to regulate mRNA export and zebrafish retinal development

Project description:MKRN2 physically interacts with GLE1 to regulate mRNA export and zebrafish retinal development

| PRJNA562493 | ENA