McCambridge, A. H. | bioRxiv Science

bioRxiv2026

The QxxR Motif of RNA Helicase Me31B Is Essential for Drosophila Female Fertility and Germline Development

The QxxR motif is evolutionarily conserved within DEAD-box RNA helicases, including Drosophila Me31B and human DDX6, which post-transcriptionally regulate gene expression during animal development. A pathogenic H372R substitution (QxHR to QxRR) in the QxxR motif of human DDX6 has been associated with various developmental defects, but how this motif contributes to DDX6-family protein function remains unclear. Here, we used Drosophila Me31B as an in vivo model to investigate the QxxR motifs developmental role. We generated a Drosophila strain carrying the corresponding H333R missense mutation in Me31B and characterized its effects on female fertility, embryonic viability, germline development, and Me31B-associated molecular pathways. The me31BH333R mutation reduced female fertility in a gene dose-dependent manner, with homozygous mutant females being sterile. Embryos from the mutant females also exhibited primordial germ cell defects. Despite these developmental phenotypes, the me31BH333R mutation did not significantly alter Me31B protein abundance, global ovarian transcriptome or proteome profiles, or representative germ plasm mRNA and protein localization. In contrast, bait-normalized IP-MS analysis revealed altered enrichment of selected Me31B-associated proteins, including increased association of known Me31B interactors Trailer hitch (Tral) and Ypsilon Schachtel (Yps). These findings establish Me31BH333R as an in vivo model for investigating the conserved QxxR motif and suggest that disruption of this motif compromises development not through broad changes in gene expression, but potentially through altered composition or regulation of Me31B-containing ribonucleoprotein complexes.

genetics↗

bioRxiv2022

An In Vivo Analysis of the Functional Motifs of DEAD-box RNA Helicase Me31B in Drosophila Fertility and Germline Development

In Drosophila germline, Me31B is a putative ATP-dependent, RNA helicase that plays role in post-transcriptional RNA regulation to ensure the correct spatial and temporal expression of the mRNAs, a process crucial for proper germline development and fertility. However, Me31Bs in vivo working mechanism remains unclear. In this study, we aim to analyze the functions of Me31Bs key domains/motifs to understand how these domains/motifs operate to fulfill the proteins overall activities. We generated Drosophila strains mutant for six important motifs including three ATPase/helicase motifs (DEAD-box, DVLARAK, and HRIGR), the N-terminal domain (N-ter), the C-terminal domain (C-ter), and a protein-binding motif (FDF motif-binding motif). In characterizing these mutants, we observed that the three ATPase/helicase motif mutations cause dominant female sterility which is associated with developmental defects in oogenesis and embryogenesis. Follow-up examination of the DVLARAK motif mutant revealed its abnormalities in germline mRNA localization and transcript level. The Me31B N-ter domain (deletion of C-ter), C-ter domain (deletion of N-ter), and mutation of FDF motif-binding motif led to a decrease in female fertility and abnormal subcellular Me31B localizations in the egg chambers. Moreover, deletion of Me31B N-ter or C-ter motif results in a decrease of Me31B protein levels in the ovaries. This study indicates that these six motifs of Me31B play different roles to contribute to Me31Bs whole-protein functions like ATPase, RNA helicase, protein stability, protein localization, and partner protein binding, which are crucial for germline development and fertility. Considering Me31B protein familys conserved presence in both Drosophila germline and soma (for example, neurons) and in other organisms such as yeast, worm, mouse, and human, the results from this study could expand our understanding of Me31B helicase familys general working mechanisms in different cell types and species.

developmental biology↗