Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila
SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two...
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oai:doaj.org-article:98c740931fee44b8b4d3bd83c87189962021-11-11T06:16:25ZTwo oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila1553-73901553-7404https://doaj.org/article/98c740931fee44b8b4d3bd83c87189962021-11-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8559932/?tool=EBIhttps://doaj.org/toc/1553-7390https://doaj.org/toc/1553-7404SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and–H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system. Author summary In the past decade, one of the important findings in the RNA splicing field has been that somatic SF3B1 mutations widely occur in many cancers. Including R625, H662, K666, K700 and E902, there are five hotspot mutation sites in the highly conserved HEAT repeats of SF3B1. Several kinds of H662 mutations have been found widely in MDS, AML, CLL and breast cancers; however, it remains unclear how these H662 mutations alter splicing and whether they have in vivo effects on development. To address these questions, in this manuscript, we first summarized the H662 mutations in human diseases and constructed two corresponding Drosophila mutant strains, sf3b1-H698D and -H698R using CRISPR-Cas9. Analyses of these two fly strains find that the two oppositely charged Sf3b1-H698 mutants are defective in development. In addition, one mutant has decreased climbing ability, whereas the other mutant has impaired immune response. Further RNA-seq allows us to find responsible genes in each mutant strain, and lariat sequencing reveals that both mutations cause aberrant selection of the intronic branch sites. Our findings provide the first in vivo evidence that Sf3b1 mutations result in defective development, and also reveal a molecular mechanism of these hotspot histidine mutations that enhance the use of cryptic branch sites to alter splicing. Importantly, we demonstrate that the H698R mutant prefers to use far upstream branch sites.Bei ZhangZhan DingLiang LiLing-Kun XieYu-Jie FanYong-Zhen XuPublic Library of Science (PLoS)articleGeneticsQH426-470ENPLoS Genetics, Vol 17, Iss 11 (2021) |
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Genetics QH426-470 Bei Zhang Zhan Ding Liang Li Ling-Kun Xie Yu-Jie Fan Yong-Zhen Xu Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila |
description |
SF3B1 mutations occur in many cancers, and the highly conserved His662 residue is one of the hotspot mutation sites. To address effects on splicing and development, we constructed strains carrying point mutations at the corresponding residue His698 in Drosophila using the CRISPR-Cas9 technique. Two mutations, H698D and H698R, were selected due to their frequent presence in patients and notable opposite charges. Both the sf3b1-H698D and–H698R mutant flies exhibit developmental defects, including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespans. Interestingly, the H698D mutant has decreased resistance to fungal infection, while the H698R mutant shows impaired climbing ability. Consistent with these phenotypes, further analysis of RNA-seq data finds altered expression of immune response genes and changed alternative splicing of muscle and neural-related genes in the two mutants, respectively. Expression of Mef2-RB, an isoform of Mef2 gene that was downregulated due to splicing changes caused by H698R, partly rescues the climbing defects of the sf3b1-H698R mutant. Lariat sequencing reveals that the two sf3b1-H698 mutations cause aberrant selection of multiple intronic branch sites, with the H698R mutant using far upstream branch sites in the changed alternative splicing events. This study provides in vivo evidence from Drosophila that elucidates how these SF3B1 hotspot mutations alter splicing and their consequences in development and in the immune system. Author summary In the past decade, one of the important findings in the RNA splicing field has been that somatic SF3B1 mutations widely occur in many cancers. Including R625, H662, K666, K700 and E902, there are five hotspot mutation sites in the highly conserved HEAT repeats of SF3B1. Several kinds of H662 mutations have been found widely in MDS, AML, CLL and breast cancers; however, it remains unclear how these H662 mutations alter splicing and whether they have in vivo effects on development. To address these questions, in this manuscript, we first summarized the H662 mutations in human diseases and constructed two corresponding Drosophila mutant strains, sf3b1-H698D and -H698R using CRISPR-Cas9. Analyses of these two fly strains find that the two oppositely charged Sf3b1-H698 mutants are defective in development. In addition, one mutant has decreased climbing ability, whereas the other mutant has impaired immune response. Further RNA-seq allows us to find responsible genes in each mutant strain, and lariat sequencing reveals that both mutations cause aberrant selection of the intronic branch sites. Our findings provide the first in vivo evidence that Sf3b1 mutations result in defective development, and also reveal a molecular mechanism of these hotspot histidine mutations that enhance the use of cryptic branch sites to alter splicing. Importantly, we demonstrate that the H698R mutant prefers to use far upstream branch sites. |
format |
article |
author |
Bei Zhang Zhan Ding Liang Li Ling-Kun Xie Yu-Jie Fan Yong-Zhen Xu |
author_facet |
Bei Zhang Zhan Ding Liang Li Ling-Kun Xie Yu-Jie Fan Yong-Zhen Xu |
author_sort |
Bei Zhang |
title |
Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila |
title_short |
Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila |
title_full |
Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila |
title_fullStr |
Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila |
title_full_unstemmed |
Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila |
title_sort |
two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in drosophila |
publisher |
Public Library of Science (PLoS) |
publishDate |
2021 |
url |
https://doaj.org/article/98c740931fee44b8b4d3bd83c8718996 |
work_keys_str_mv |
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