AnIn VivoAnalysis of the Functional Motifs of DEAD-box RNA Helicase Me31B inDrosophilaFertility and Germline Development

Abstract

AbstractInDrosophilagermline, 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, Me31B’sin vivoworking mechanism remains unclear. In this study, we aim to analyze the functions of Me31B’s key domains/motifs to understand how these domains/motifs operate to fulfill the protein’s overall activities. We generatedDrosophilastrains 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 Me31B’s 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 family’s conserved presence in bothDrosophilagermline 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 family’s general working mechanisms in different cell types and species.