Refine
Year of publication
- 2017 (4)
Document Type
- Article (4)
Has Fulltext
- yes (4)
Is part of the Bibliography
- no (4)
Keywords
- Biochemistry (1)
- RNA Biology (1)
Institute
- Exzellenzcluster Makromolekulare Komplexe (4) (remove)
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.
"Ästhetisch ist, was hilft"
(2017)
We have reported previously that Short Interspersed Degenerate Retroposons of the SIDER2 subfamily, largely located within 3'UTRs of Leishmania transcripts, promote rapid turnover of mRNAs through endonucleolytic cleavage within the highly conserved second tandem 79-nt hallmark sequence (79-nt SII). Here, we used site-directed mutagenesis and in silico RNA structural studies to delineate the cis-acting requirements within 79-nt SII for cleavage and mRNA degradation. The putative cleavage site(s) and other nucleotides predicted to alter the RNA secondary structure of 79-nt SII were either deleted or mutated and their effect on mRNA turnover was monitored using a gene reporter system. We found that short deletions of 8-nt spanning the two predicted cleavage sites block degradation of SIDER2-containing transcripts, leading to mRNA accumulation. Furthermore, single or double substitutions of the dinucleotides targeted for cleavage as well as mutations altering the predicted RNA secondary structure encompassing both cleavage sites also prevent mRNA degradation, confirming that these dinucleotides are the bona fide cleavage sites. In line with these results, we show that stage-regulated SIDER2 inactivation correlates with the absence of endonucleolytic cleavage. Overall, these data demonstrate that both cleavage sites within the conserved 79-nt SII as well as RNA folding in this region are essential for SIDER2-mediated mRNA decay, and further support that SIDER2-harboring transcripts are targeted for degradation by endonucleolytic cleavage.
A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei. Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome.