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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.

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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.

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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.

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Control of gene expression by Ott1/RBM15 protein

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Transcriptome maps of mRNP biogenesis factors define pre-mRNA recognition

Project description:Biogenesis of eukaryotic messenger ribonucleoprotein complexes (mRNPs) involves the synthesis, splicing, and 3M-bM-^@M-^Y-processing of pre-mRNA, and the assembly of mature mRNPs for nuclear export. We mapped 23 mRNP biogenesis factors onto the newly synthesized yeast transcriptome, providing ~10^5-10^6 high-confidence RNA interaction sites per factor. PAR-CLIP data of 23 mRNP biogenesis factors in Saccharomyces cerevisiae

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Epstein-Barr virus protein EB2 stimulates translation initiation of mRNAs through direct interactions with both PABP and eIF4G

Project description:Epstein-Barr virus (EBV) expresses several mRNAs produced from intronless genes that could be unfavorably translated in front of cellular spliced mRNAs. To overpass these limitations, the virus encodes a RNA-binding protein (RBP), EB2, which is already known to facilitate nuclear mRNAs export and to increase their translational yield. Here we show that EB2 binds both nuclear and cytoplasmic cap-binding complexes (respectively, CBC and eIF4F), and the poly(A)-binding protein (PABP) to enhance translation initiation of a given mRNP. Interestingly, such effect can be obtained only if EB2 was initially bound to the native mRNPs in the nucleus. We also demonstrate that the EB2-eIF4F-PABP association renders the translation of these mRNPs less sensitive to inhibitors of initiation. Taken together, our new data suggest that EB2 binds and stabilizes cap-binding complexes to increase mRNP translation and demonstrate the importance of the mRNP assembly process in the nucleus to promote translation in the cytoplasm.

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The SR-like protein Npl3 transfers mRNP components from the transcription site onto the mRNA

Project description:In order to determine specifically the RNA-binding function of proteins involved in nuclear mRNP assembly, we first determined the amino acids involved in RNA-binding by RNPXL. We identified about 100 amino acids cross-linked to RNA in vivo in the nuclear mRNP components Npl3, Nab2, Tho1, Mex67-Mtr2 and the TREX complex. Second, we can now specifically elucidate the function of the RNA-binding activity of these proteins by mutation of the identified amino acids. Npl3 is an SR-like protein with functions in transcription elongation, splicing, 3’ end processing, mRNP assembly and nuclear mRNA export. The middle part of Npl3 consists of two RNA recognition motif (RRM) domains, RRM1 and RRM2, connected by an eight amino acid long flexible linker. In order to analyze the function of the RNA-binding activity of Npl3, we mutated several amino acids that cross-linked to RNA. We generated three npl3 mutants, one in the RRM1, one in the linker and a third in the RRM2 domain, and elucidated the functional consequences of these mutations. Interestingly, these three npl3 mutants show different phenotypes. Thus, abrogation of mRNA-binding in different regions of Npl3 has different functional outcomes. Furthermore, analysis of the npl3-Linker mutant revealed a novel function of Npl3. Npl3 functions in the transfer of nuclear mRNP components from the site of transcription onto the mRNA. Taken together, we identify the in vivo RNA-binding sites of nuclear mRNA-binding proteins involved in mRNP assembly and nuclear mRNA export. In addition, we show that abrogation of RNA-binding in different regions of the protein Npl3 has specific and surprisingly different functional consequences. Furthermore, we uncovered a novel function of Npl3 in nuclear mRNP assembly.

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