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The RNA-binding activity of the <i>Drosophila</i> Brat protein is necessary for viability and mRNA regulation.

Overview

Authors: Robert P Connacher1, Yichao Hu2, Richard Roden1, Julia Toledo1, Anna DesMarais1, Michael O’Connor3, Howard D Lipshitz2, Aaron C Goldstrohm1
ORCID iDs: Robert P Connacher, Aaron C Goldstrohm
  1. Department of Biochemistry, Molecular Biology, & Biophysics, University of Minnesota, Minneapolis, MN, USA
  2. Department of Molecular Genetics, University of Toronto, Toronto, Canada
  3. Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA
Institutions: University of Minnesota (United States); University of Toronto (Canada)
Journal: RNA biology, volume 23, issue 1, pages 1-19
Dates: accepted 26 May 2026; published online 28 May 2026; in print December 2026
Type: Research article · Language: English
License: CC BY
Identifiers: DOI 10.1080/15476286.2026.2682069 · PMID 42206510 · PMCID PMC13245067 · OpenAlex W7162659848
Open access: gold, a free copy (OpenAlex)
Status: data only
Categories: drosophila (organism)
Methods: Spectral & time-frequency, Statistics, Evoked potentials, fMRI & imaging
Keywords: RNA-binding protein, development, neural stem cells, embryogenesis, CRISPR
MeSH: DNA-Binding Proteins*, Drosophila melanogaster*, Drosophila Proteins*, RNA, Messenger*, RNA-Binding Proteins*, Alleles, Animals, Binding Sites, Gene Expression Regulation, Developmental, Mutation, Protein Binding (* major topic)
Topic: Developmental Biology and Gene Regulation (Molecular Biology, Biochemistry, Genetics and Molecular Biology), according to OpenAlex
Funding: National Institutes of Health (GM105707, GM145835); National Institute of General Medical Sciences; NIGMS NIH HHS (R01 GM145835, R01 GM105707); Minneapolis Foundation (Edith Walters Jones and Robert Jones Fellowship); Canadian Institutes of Health Research (PJT-190124); Edith Walters Jones and Robert Jones Fellowship
Citations: not cited yet (Europe PMC); 74 references in the paper

Abstract

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’s 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 brat 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 brat null and hypomorphic alleles. Lethal phase analysis revealed death predominantly during late larval and pupal stages, consistent with known brat alleles. Consistent with the namesake brat 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 brat 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 an essential molecular activity of Brat and that post-transcriptional regulation of Brat target mRNAs underlies its critical roles across development.

Reproduced under the paper's license (CC BY), from the paper cited above.

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Data

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The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Reproduced under the paper's license (CC BY), from the paper cited above.

Versions

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Version 1, 27 September 2026: the first record

Recorded: type, language, journal, volume, issue, pages, dates, 8 authors, 5 keywords, 11 MeSH terms, 6 funders, 70 references.

Cite

This paper

Connacher, R. P., Hu, Y., Roden, R., Toledo, J., DesMarais, A., O’Connor, M., Lipshitz, H. D., & Goldstrohm, A. C. (2026). The RNA-binding activity of the <i>Drosophila</i> Brat protein is necessary for viability and mRNA regulation. RNA biology, 23(1), 1-19. https://doi.org/10.1080/15476286.2026.2682069

BibTeX

@article{connacher2026rna,
author = {Connacher, Robert P and Hu, Yichao and Roden, Richard and Toledo, Julia and DesMarais, Anna and O’Connor, Michael and Lipshitz, Howard D and Goldstrohm, Aaron C},
title = {{The RNA-binding activity of the \<i\>Drosophila\</i\> Brat protein is necessary for viability and mRNA regulation}},
journal = {RNA biology},
year = {2026},
month = jun,
volume = {23},
number = {1},
pages = {1--19},
publisher = {Taylor \& Francis},
issn = {1547-6286},
doi = {10.1080/15476286.2026.2682069},
url = {https://doi.org/10.1080/15476286.2026.2682069},
pmid = {42206510},
pmcid = {PMC13245067}
}

RIS

TY - JOUR
AU - Connacher, Robert P
AU - Hu, Yichao
AU - Roden, Richard
AU - Toledo, Julia
AU - DesMarais, Anna
AU - O’Connor, Michael
AU - Lipshitz, Howard D
AU - Goldstrohm, Aaron C
TI - The RNA-binding activity of the <i>Drosophila</i> Brat protein is necessary for viability and mRNA regulation
T2 - RNA biology
J2 - RNA Biol
PY - 2026
DA - 2026/06/05
VL - 23
IS - 1
SP - 1
EP - 19
SN - 1547-6286
PB - Taylor & Francis
DO - 10.1080/15476286.2026.2682069
UR - https://doi.org/10.1080/15476286.2026.2682069
LA - en
ER -

CSL-JSON

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