A time-resolved framework for the recruitment of mRNP processing and assembly factors to a site of transcription
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ABSTRACT:
PROVIDER: PRJNA1246517 | ENA |
REPOSITORIES: ENA
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Project description:Processing and packaging of messenger ribonucleoprotein (mRNP) particles involve complex, coordinated interactions between nascent transcripts, RNA binding proteins (RBPs), and other associated factors. Despite the critical role of co-transcriptional mRNP assembly in gene expression, the temporal dynamics of this process are not well understood. Here, a live cell imaging assay is reported in S. cerevisiae to quantitatively detect recruitment of endogenous fluorescently tagged proteins to a transcriptionally active locus. Protein recruitment to an inducible integrated gene array composed of twenty-five transcriptional units is detected by colocalization with lacO repeats. Using arrays with two different promoters and the same coding sequence (GFA1), arrival times for a variety of mRNP processing and assembly factors were quantified. These analyses revealed Yra1, Cbp80, and Yhs7 as pioneering assembly factors. Notably, Yra1 recruitment occurs independently of the THO complex, with early localization supported by Cbp80 and the RNA recognition motif (RRM) of Yra1. Altogether, this work establishes the first comprehensive temporal framework for understanding protein recruitment during co-transcriptional mRNP assembly, providing mechanistic insights into the dependencies of Yra1 recruitment.
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Project description:RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate several functions at once and likely impact the structural integrity of the large mRNP assemblies, these proteins often function in. Thus, the function of the RNA-binding activity of RBPs is often unknown. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric (MS) analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components. For functional analysis, we chose Npl3, for which we identified crosslinks in its two RNA recognition motifs (RRMs) as well as in the flexible linker connecting the two. Both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in each of these three regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutant strongly impairs recruitment of some mRNP components to chromatin and the incorporation of other mRNP components into nuclear mRNPs, establishing a function of Npl3 in mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of a nuclear mRNP component, an approach that can be applied to many RBPs in any RNA metabolic process.
2023-03-11 | PXD035153 | Pride
Project description:Assessing the function of the RNA-binding activity of RNA-binding proteins (RBPs) is challenging. Here, Keil et al. use a targeted XL-ms approach to identify RNA-binding sites in components of nuclear mRNPs in S. cerevisiae. The analysis of RNA-binding mutants of one selected example, the SR-like protein Npl3, uncovers two novel functions of Npl3, namely in the recruitment of mRNP components to transcribed protein-coding genes and their transfer onto the mRNA. This data set applies label-free protein quantification to assess protein abundancies in NPL3 or CBC2 affinity purification experiments from cellular lysates of NPL3-WT and NPL3-P196D/A197D double-mutant expressing cells.
2023-03-11 | PXD034656 | Pride
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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2023-04-30 | E-MTAB-12378 | biostudies-arrayexpress
Project description:RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.
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Project description:Biogenesis of eukaryotic messenger ribonucleoprotein complexes (mRNPs) involves the synthesis, splicing, and 3’-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.
2014-07-23 | GSE59676 | GEO