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Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster

Methylphosphate Capping Enzyme (MEPCE) monomethylates the gamma phosphate at the 5’ end of the 7SK noncoding RNA, a modification thought to protect 7SK from degradation. 7SK serves as a scaffold for assembly of a snRNP complex that inhibits transcription by sequestering the positive elongation facto...

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Autores principales: Palumbo, Ryan J, Hanes, Steven D
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10274667/
https://www.ncbi.nlm.nih.gov/pubmed/37333392
http://dx.doi.org/10.1101/2023.06.01.543302
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author Palumbo, Ryan J
Hanes, Steven D
author_facet Palumbo, Ryan J
Hanes, Steven D
author_sort Palumbo, Ryan J
collection PubMed
description Methylphosphate Capping Enzyme (MEPCE) monomethylates the gamma phosphate at the 5’ end of the 7SK noncoding RNA, a modification thought to protect 7SK from degradation. 7SK serves as a scaffold for assembly of a snRNP complex that inhibits transcription by sequestering the positive elongation factor P-TEFb. While much is known about the biochemical activity of MEPCE in vitro, little is known about its functions in vivo, or what roles— if any—there are for regions outside the conserved methyltransferase domain. Here, we investigated the role of Bin3, the Drosophila ortholog of MEPCE, and its conserved functional domains in Drosophila development. We found that bin3 mutant females had strongly reduced rates of egg-laying, which was rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 promotes fecundity by repressing P-TEFb. bin3 mutants also exhibited neuromuscular defects, analogous to a patient with MEPCE haploinsufficiency. These defects were also rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 and MEPCE have conserved roles in promoting neuromuscular function by repressing P-TEFb. Unexpectedly, we found that a Bin3 catalytic mutant (Bin3(Y795A)) could still bind and stabilize 7SK and rescue all bin3 mutant phenotypes, indicating that Bin3 catalytic activity is dispensable for 7SK stability and snRNP function in vivo. Finally, we identified a metazoan-specific motif (MSM) outside of the methyltransferase domain and generated mutant flies lacking this motif (Bin3(ΔMSM)). Bin3(ΔMSM) mutant flies exhibited some—but not all—bin3 mutant phenotypes, suggesting that the MSM is required for a 7SK-independent, tissue-specific function of Bin3.
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spelling pubmed-102746672023-06-17 Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster Palumbo, Ryan J Hanes, Steven D bioRxiv Article Methylphosphate Capping Enzyme (MEPCE) monomethylates the gamma phosphate at the 5’ end of the 7SK noncoding RNA, a modification thought to protect 7SK from degradation. 7SK serves as a scaffold for assembly of a snRNP complex that inhibits transcription by sequestering the positive elongation factor P-TEFb. While much is known about the biochemical activity of MEPCE in vitro, little is known about its functions in vivo, or what roles— if any—there are for regions outside the conserved methyltransferase domain. Here, we investigated the role of Bin3, the Drosophila ortholog of MEPCE, and its conserved functional domains in Drosophila development. We found that bin3 mutant females had strongly reduced rates of egg-laying, which was rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 promotes fecundity by repressing P-TEFb. bin3 mutants also exhibited neuromuscular defects, analogous to a patient with MEPCE haploinsufficiency. These defects were also rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 and MEPCE have conserved roles in promoting neuromuscular function by repressing P-TEFb. Unexpectedly, we found that a Bin3 catalytic mutant (Bin3(Y795A)) could still bind and stabilize 7SK and rescue all bin3 mutant phenotypes, indicating that Bin3 catalytic activity is dispensable for 7SK stability and snRNP function in vivo. Finally, we identified a metazoan-specific motif (MSM) outside of the methyltransferase domain and generated mutant flies lacking this motif (Bin3(ΔMSM)). Bin3(ΔMSM) mutant flies exhibited some—but not all—bin3 mutant phenotypes, suggesting that the MSM is required for a 7SK-independent, tissue-specific function of Bin3. Cold Spring Harbor Laboratory 2023-06-08 /pmc/articles/PMC10274667/ /pubmed/37333392 http://dx.doi.org/10.1101/2023.06.01.543302 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Palumbo, Ryan J
Hanes, Steven D
Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster
title Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster
title_full Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster
title_fullStr Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster
title_full_unstemmed Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster
title_short Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster
title_sort catalytic activity of the bin3/mepce methyltransferase domain is dispensable for 7sk snrnp function in drosophila melanogaster
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10274667/
https://www.ncbi.nlm.nih.gov/pubmed/37333392
http://dx.doi.org/10.1101/2023.06.01.543302
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