A molecular link between SR protein dephosphorylation and mRNA export.
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ABSTRACT:
SUBMITTER: Huang Y
PROVIDER: S-EPMC470732 | biostudies-other | 2004 Jun
REPOSITORIES: biostudies-other
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Project description:Nuclear export factor 1 (NXF1) exports mRNA to the cytoplasm after recruitment to mRNA by specific adaptor proteins. How and why cells use numerous different export adaptors is poorly understood. Here we critically evaluate members of the SR protein family (SRSF1-7) for their potential to act as NXF1 adaptors that couple pre-mRNA processing to mRNA export. Consistent with this proposal, >1000 endogenous mRNAs required individual SR proteins for nuclear export in vivo. To address the mechanism, transcriptome-wide RNA-binding profiles of NXF1 and SRSF1-7 were determined in parallel by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP). Quantitative comparisons of RNA-binding sites showed that NXF1 and SR proteins bind mRNA targets at adjacent sites, indicative of cobinding. SRSF3 emerged as the most potent NXF1 adaptor, conferring sequence specificity to RNA binding by NXF1 in last exons. Interestingly, SRSF3 and SRSF7 were shown to bind different sites in last exons and regulate 3' untranslated region length in an opposing manner. Both SRSF3 and SRSF7 promoted NXF1 recruitment to mRNA. Thus, SRSF3 and SRSF7 couple alternative splicing and polyadenylation to NXF1-mediated mRNA export, thereby controlling the cytoplasmic abundance of transcripts with alternative 3' ends.
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Project description:SR proteins function in nuclear pre-mRNA processing, mRNA export, and translation. To investigate their cellular dynamics, we developed a quantitative assay, which detects differences in nucleocytoplasmic shuttling among seven canonical SR protein family members. As expected, SRSF2 and SRSF5 shuttle poorly in HeLa cells but surprisingly display considerable shuttling in pluripotent murine P19 cells. Combining individual-resolution cross-linking and immunoprecipitation (iCLIP) and mass spectrometry, we show that elevated arginine methylation of SRSF5 and lower phosphorylation levels of cobound SRSF2 enhance shuttling of SRSF5 in P19 cells by modulating protein-protein and protein-RNA interactions. Moreover, SRSF5 is bound to pluripotency-specific transcripts such as Lin28a and Pou5f1/Oct4 in the cytoplasm. SRSF5 depletion reduces and overexpression increases their cytoplasmic mRNA levels, suggesting that enhanced mRNA export by SRSF5 is required for the expression of pluripotency factors. Remarkably, neural differentiation of P19 cells leads to dramatically reduced SRSF5 shuttling. Our findings indicate that posttranslational modification of SR proteins underlies the regulation of their mRNA export activities and distinguishes pluripotent from differentiated cells.
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Project description:Cell growth, differentiation, and commitment to a restricted lineage are guided by a timely expressed set of growth factor/cytokine receptors and their down-stream transcription factor genes. Transcriptional control mechanisms of gene expression during differentiation have been mainly studied by focusing on the cis- and trans-elements in promoters however, the role of mRNA export machinery during differentiation has not been adequately examined. THO (Suppressors of the transcriptional defects of hpr1 delta by overexpression) complex 5 (THOC5) is a member of THO complex which is a subcomplex of the transcription/export complex (TREX). THOC5 is evolutionarily conserved in higher eukaryotes, however the exact roles of THOC5 in transcription and mRNA export are still unclear. In this review, we focus on recently uncovered aspects of the role of THOC5 in signal transduction induced by extracellular stimuli. THOC5 is phosphorylated by several protein kinases at multiple residues upon extracellular stimuli. These include stimulation with growth factors/cytokines/chemokines, or DNA damage reagents. Furthermore, THOC5 is a substrate for several oncogenic tyrosine kinases, suggesting that THOC5 may be involved in cancer development. Recent THOC5 knockout mouse data reveal that THOC5 is an essential element in the maintenance of stem cells and growth factor/cytokine-mediated differentiation/proliferation. Furthermore, depletion of THOC5 influences less than 1% of total mRNA export in the steady state, however it influences more than 90% of growth factor/cytokine induced genes. THOC5, thereby contributes to the 3' processing and/or export of immediate-early genes induced by extracellular stimuli. These studies bring new insight into the link between the mRNA export complex and immediate-early gene response. The data from these studies also suggest that THOC5 may be a useful tool for studying stem cell biology, for modifying the differentiation processes and for cancer therapy.
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Project description:Viruses employ multiple strategies to inhibit host mRNA nuclear export. Distinct to the generally nonselective inhibition mechanisms, ORF10 from gammaherpesviruses inhibits mRNA export in a transcript-selective manner by interacting with Rae1 (RNA export 1) and Nup98 (nucleoporin 98). We now report the structure of ORF10 from MHV-68 (murine gammaherpesvirus 68) bound to the Rae1-Nup98 heterodimer, thereby revealing detailed intermolecular interactions. Structural and functional assays highlight that two highly conserved residues of ORF10, L60 and M413, play critical roles in both complex assembly and mRNA export inhibition. Interestingly, although ORF10 occupies the RNA-binding groove of Rae1-Nup98, the ORF10-Rae1-Nup98 ternary complex still maintains a comparable RNA-binding ability due to the ORF10-RNA direct interaction. Moreover, mutations on the RNA-binding surface of ORF10 disrupt its function of mRNA export inhibition. Our work demonstrates the molecular mechanism of ORF10-mediated selective inhibition and provides insights into the functions of Rae1-Nup98 in regulating host mRNA export.
| S-EPMC7604486 | biostudies-literature
Project description:Nuclear export of mRNA requires several key mRNA-binding proteins that recognize and remodel the mRNA and target it for export via interactions with the nuclear pore complex. In Saccharomyces cerevisiae, the shuttling heterogeneous nuclear ribonucleoprotein, Nab2, which is essential for mRNA export, specifically recognizes poly(A) RNA and binds to the nuclear pore-associated protein, myosin-like protein 1 (Mlp1), which functions in mRNA export and quality control. Specifically, the N-terminal domain of Nab2 (Nab2-N; residues 1-97) interacts directly with the C-terminal globular domain of Mlp1 (CT-Mlp1: residues 1490-1875). Recent structural and binding studies focused on Nab2-N have shown that Nab2-N contains a hydrophobic patch centered on Phe(73) that is critical for interaction with Mlp1. Engineered amino acid changes within this patch disrupt the Nab2/Mlp1 interaction in vitro. Given the importance of Nab2 and Mlp1 to mRNA export, we have examined the Nab2/Mlp1 interaction in greater detail and analyzed the functional consequences of disrupting the interaction in vivo. We find that the Nab2-binding domain of Mlp1 (Mlp1-NBD) maps to a 183-residue region (residues 1586-1768) within CT-Mlp1, binds directly to Nab2 with micromolar affinity, and confers nuclear accumulation of poly(A) RNA. Furthermore, we show that cells expressing a Nab2 F73D mutant that cannot interact with Mlp1 exhibit nuclear accumulation of poly(A) RNA and that this nab2 F73D mutant genetically interacts with alleles of two essential mRNA export genes, MEX67 and YRA1. These data provide in vivo evidence for a model of mRNA export in which Nab2 is important for targeting mRNAs to the nuclear pore for export.
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Project description:Selective movement of proteins between the nucleus and the cytoplasm is a regulatory mechanism exploited extensively by the eukaryotic cell. We have identified the evolutionarily conserved Sac3 protein, which was implicated previously in the regulation of mitosis [Bauer, A. & Kölling, R. (1996) J. Cell Sci. 109, 1575-1583] as a novel mediator of nuclear protein export. We show that Sac3p is localized to the nuclear pore, where it interacts with nucleoporins. Loss of SAC3 function results in a block in nuclear export of a nuclear export signal-containing reporter protein. Our results also demonstrate that SAC3 interacts genetically with the nuclear protein export factors Crm1p/Xpo1p and Yrb2p. Taken together, these data indicate a link between nuclear protein export and transition through the cell cycle.
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Project description:Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.
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Project description:BackgroundEvidence in diverse organisms suggests that codon optimality is a major determinant of mRNA translation and degradation. Codon optimality is thought to act by modulating the efficiency of ribosome elongation. In Saccharomyces cerevisiae, a recent study has identified 17 adjacent codon pairs that mediate strong inhibition of translation elongation. However, relationships between the inhibitory codon pairs and other aspects of gene expression are unknown.ResultsTo gain insights into how the inhibitory codon pairs may affect aspects of gene expression, we utilized existing datasets to conduct genome-scale analyses in S. cerevisiae. Our analysis revealed the following points. First, the inhibitory codon pairs are significantly associated with faster mRNA decay. The association is not solely due to the content of nucleotides, individual codons, or dipeptides encoded by the inhibitory codon pairs. Second, the inhibitory codon pairs cannot fully explain the previously known relationship of codon optimality with mRNA stability, suggesting that optimality of individual codons and properties of adjacent codon pairs both contribute to gene regulation. Finally, although the inhibitory codon pairs are associated with slower mRNA synthesis and protein instability, the associations can be attributed to usage bias in individual codons.ConclusionsThis study suggests an association of inhibitory codon pairs with mRNA stability and thus another layer of complexity in the codon-mediated gene regulation.
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Project description:The breast cancer tumor suppressor BRCA2-interacting protein, DSS1, and its homologs are critical for DNA recombination in eukaryotic cells. We found that Dss1p, along with Mlo3p and Uap56p, Schizosaccharomyces pombe homologs of two messenger RNA (mRNA) export factors of the NXF-NXT pathway, is required for mRNA export in S. pombe. Previously, we showed that the nuclear pore-associated Rae1p is an essential mRNA export factor in S. pombe. Here, we show that Dss1p and Uap56p function by linking mRNA adapter Mlo3p to Rae1p for targeting mRNA-protein complex (mRNP) to the proteins of the nuclear pore complex (NPC). Dss1p preferentially recruits to genes in vivo and interacts with -FG (phenylalanine glycine) nucleoporins in vivo and in vitro. Thus, Dss1p may function at multiple steps of mRNA export, from mRNP biogenesis to their targeting and translocation through the NPC.
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Project description:Alternative polyadenylation (APA) produces mRNA isoforms with different 3' UTR lengths. Previous studies indicated that 3' end processing and mRNA export are intertwined in gene regulation. Here, we show that mRNA export factors generally facilitate usage of distal cleavage and polyadenylation sites (PASs), leading to long 3' UTR isoform expression. By focusing on the export receptor NXF1, which exhibits the most potent effect on APA in this study, we reveal several gene features that impact NXF1-dependent APA, including 3' UTR size, gene size, and AT content. Surprisingly, NXF1 downregulation results in RNA polymerase II (Pol II) accumulation at the 3' end of genes, correlating with its role in APA regulation. Moreover, NXF1 cooperates with CFI-68 to facilitate nuclear export of long 3' UTR isoform with UGUA motifs. Together, our work reveals important roles of NXF1 in coordinating transcriptional dynamics, 3' end processing, and nuclear export of long 3' UTR transcripts, implicating NXF1 as a nexus of gene regulation.
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