No abstract

In the Drosophila germline, Me31B is a putative ATP-dependent, RNA helicase that plays an important role in post-transcriptional RNA regulation to ensure mRNA′s correct spatial and temporal expression, a process crucial for proper germline development and fertility. However, Me31B′s in vivo working 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 generated mutant Drosophila strains 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 examinations of the DVLARAK motif mutant revealed 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 the FDF motif-binding motif led to a decrease in female fertility and abnormal subcellular Me31B localizations within the egg chambers. Moreover, deletion of Me31B′s N-ter or C-ter motif decreases Me31B protein levels in the ovaries. This study indicates that these six motifs of Me31B play distinct roles in contributing to Me31B′s whole-protein functions such as ATPase, RNA helicase, protein stability, protein localization, and partner protein binding, crucial for germline development and fertility. Considering the Me31B protein family′s conserved presence in both Drosophila germline and soma and in other eukaryotes such as yeast, worms, mice, and humans, the results from this study could expand our understanding of Me31B family helicases′ general working mechanisms in different cell types and species.

Me31B/DDX6 is a DEAD-box family RNA helicase playing roles in posttranscriptional RNA regulation in different cell types and species. Despite the known motifs/domains of Me31B, the in vivo functions of the motifs remain unclear. Here, we used the Drosophila germline as a model and used CRISPR to mutate the key Me31B motifs/domains: helicase domain, N-terminal domain, C-terminal domain and FDF-binding motif. Then, we performed screening characterization on the mutants and report the effects of the mutations on the Drosophila germline, on processes such as fertility, oogenesis, embryo patterning, germline mRNA regulation and Me31B protein expression. The study indicates that the Me31B motifs contribute different functions to the protein and are needed for proper germline development, providing insights into the in vivo working mechanism of the helicase.

No abstract

The co-packaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to post-transcriptionally regulate mRNAs that function in germline development and maintenance. In D. melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates that contain multiple transcripts from a specific gene. Nucleated by Oskar (Osk), homotypic clusters in D. melanogaster are generated through a stochastic seeding and self-recruitment process that requires the 3'UTR of germ granule mRNAs. Interestingly, the 3'UTR belonging to germ granule mRNAs, such as nanos (nos), have considerable sequence variations among Drosophila species. Thus, we hypothesized that evolutionary changes in the 3'UTR influences germ granule development. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that homotypic clustering is a conserved developmental process used to enrich germ granule mRNAs. Additionally, we discovered that the number of transcripts found in nos and/or pgc clusters could vary significantly among species. By integrating biological data with computational modeling, we determined that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels, and/or homotypic clustering efficacy. Finally, we found that the nos 3'UTR from different species can alter the efficacy of nos homotypic clustering, resulting in germ granules with reduced nos accumulation. Our findings highlight the impact that evolution has on the development of germ granules and may provide insight into processes that modify the content of other classes of biomolecular condensates.