{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T10:46:24Z","timestamp":1785408384492,"version":"3.56.0"},"posted":{"date-parts":[[2026,2,27]]},"group-title":"Molecular Biology","reference-count":71,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T00:00:00Z","timestamp":1772150400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R01 GM145835"],"award-info":[{"award-number":["R01 GM145835"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 GM105707"],"award-info":[{"award-number":["R01 GM105707"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["PJT-190124"],"award-info":[{"award-number":["PJT-190124"]}],"id":[{"id":"https:\/\/ror.org\/01gavpb45","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,2,27]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Brain tumor (Brat) is a Drosophila TRIM-NHL protein required for embryogenesis and neural stem cell differentiation. Although structural and biochemical studies established that the Brat NHL domain specifically binds RNA, the in vivo requirement for this activity has not been directly tested. Here, we used structure-guided mutagenesis and genome engineering to determine whether RNA recognition is essential for Brat function during development. The direct interaction between Brat\u2019s NHL domain and RNA containing Brat Binding Sites (BBS) can be abolished by alanine substitution of three separate residues on the NHL surface. We introduced these point mutations into the endogenous\n                  <jats:italic>brat<\/jats:italic>\n                  locus by CRISPR-mediated Scarless Gene Editing to generate three independent RNA-binding defective mutant (RBDmt) alleles. Complementation tests demonstrated that each allele behaves as a strong loss-of-function mutation: homozygotes and hemizygotes are inviable, and RBDmt alleles fail to complement classical\n                  <jats:italic>brat<\/jats:italic>\n                  null and hypomorphic alleles. Lethal phase analysis revealed death predominantly during late larval and pupal stages, consistent with known\n                  <jats:italic>brat<\/jats:italic>\n                  alleles. Consistent with the namesake\n                  <jats:italic>brat<\/jats:italic>\n                  phenotype, RBDmt larval brains exhibited widespread expression of neuroblast markers and a marked reduction of neuronal differentiation. In embryos, these alleles failed to complement female sterile\n                  <jats:italic>brat<\/jats:italic>\n                  alleles and recapitulated characteristic abdominal segmentation defects. Finally, RT-qPCR showed increased expression of endogenous Brat target mRNAs in mutant larvae, consistent with loss of Brat-mediated repression. Together, these results demonstrate that direct RNA binding is the essential molecular activity of Brat and that post-transcriptional regulation of Brat target mRNAs underlies its critical roles across development.\n                <\/jats:p>","DOI":"10.64898\/2026.02.27.708583","type":"posted-content","created":{"date-parts":[[2026,2,28]],"date-time":"2026-02-28T03:55:18Z","timestamp":1772250918000},"source":"Crossref","is-referenced-by-count":0,"title":["RNA-binding is the essential biological function of the\n                  <i>Drosophila<\/i>\n                  protein Brat"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4560-3852","authenticated-orcid":false,"given":"Robert P.","family":"Connacher","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, & Biophysics, University of Minnesota, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-2724-0253","authenticated-orcid":false,"given":"Yichao","family":"Hu","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics, University of Toronto, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard","family":"Roden","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, & Biophysics, University of Minnesota, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Toledo","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, & Biophysics, University of Minnesota, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"DesMarais","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, & Biophysics, University of Minnesota, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"O\u2019Connor","sequence":"additional","affiliation":[{"name":"Department of Genetics, Cell Biology, and Development, University of Minnesota, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Howard D.","family":"Lipshitz","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics, University of Toronto, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1867-8763","authenticated-orcid":false,"given":"Aaron C.","family":"Goldstrohm","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, & Biophysics, University of Minnesota, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026030215250548000_2026.02.27.708583v1.1","doi-asserted-by":"crossref","first-page":"e1620","DOI":"10.1002\/wrna.1620","article-title":"Molecular and biological functions of TRIM-NHL RNA-binding proteins","volume":"12","year":"2021","journal-title":"Wiley Interdiscip Rev RNA"},{"key":"2026030215250548000_2026.02.27.708583v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-03920-7"},{"key":"2026030215250548000_2026.02.27.708583v1.3","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-015-0659-4"},{"key":"2026030215250548000_2026.02.27.708583v1.4","doi-asserted-by":"crossref","first-page":"1206","DOI":"10.1016\/j.celrep.2015.09.068","article-title":"The Crystal Structure of the NHL Domain in Complex with RNA Reveals the Molecular Basis of Drosophila Brain-Tumor-Mediated Gene Regulation","volume":"13","year":"2015","journal-title":"Cell reports"},{"key":"2026030215250548000_2026.02.27.708583v1.5","doi-asserted-by":"publisher","DOI":"10.15252\/embr.201744188"},{"key":"2026030215250548000_2026.02.27.708583v1.6","doi-asserted-by":"crossref","first-page":"12669","DOI":"10.1093\/nar\/gkae810","article-title":"The TRIM-NHL RNA-binding protein Brain Tumor coordinately regulates expression of the glycolytic pathway and vacuolar ATPase complex","volume":"52","year":"2024","journal-title":"Nucleic Acids Res"},{"key":"2026030215250548000_2026.02.27.708583v1.7","doi-asserted-by":"publisher","DOI":"10.1101\/gad.236513.113"},{"key":"2026030215250548000_2026.02.27.708583v1.8","doi-asserted-by":"publisher","DOI":"10.1101\/gad.870801"},{"key":"2026030215250548000_2026.02.27.708583v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.cub.2006.08.093"},{"key":"2026030215250548000_2026.02.27.708583v1.10","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.16955"},{"key":"2026030215250548000_2026.02.27.708583v1.11","doi-asserted-by":"publisher","DOI":"10.1038\/nature13007"},{"key":"2026030215250548000_2026.02.27.708583v1.12","doi-asserted-by":"crossref","unstructured":"Vastenhouw NL , Cao WX , Lipshitz HD . 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