Germ Granule Evolution Provides Mechanistic Insight into Drosophila Germline Development.

The copackaging of mRNAs into biomolecular condensates called germ granules is a conserved strategy to posttranscriptionally regulate germline mRNAs. In Drosophila melanogaster, mRNAs accumulate in germ granules by forming homotypic clusters, aggregates containing multiple transcripts from the same gene. Nucleated by Oskar (Osk), homotypic clusters are generated through a stochastic seeding and self-recruitment process that requires the 3' untranslated region (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 and we hypothesized that this diversity influences homotypic clustering. To test our hypothesis, we investigated the homotypic clustering of nos and polar granule component (pgc) in four Drosophila species and concluded that clustering is a conserved process used to enrich germ granule mRNAs. However, we discovered germ granule phenotypes that included significant changes in the abundance of transcripts present in species' homotypic clusters, which also reflected diversity in the number of coalesced primordial germ cells within their embryonic gonads. By integrating biological data with computational modeling, we found that multiple mechanisms underlie naturally occurring germ granule diversity, including changes in nos, pgc, osk levels and/or homotypic clustering efficacy. Furthermore, we demonstrated how the nos 3' UTR from different species influences nos clustering, causing granules to have ∼70% less nos and increasing the presence of defective primordial germ cells. Our results highlight the impact that evolution has on germ granules, which should provide broader insight into processes that modify compositions and activities of other classes of biomolecular condensate.

Mutational analysis of the functional motifs of the DEAD-box RNA helicase Me31B/DDX6 in Drosophila germline development.

Me31B/DDX6 is a DEAD-box family RNA helicase playing roles in post-transcriptional 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.

Evolutionary changes in germ granule mRNA content are driven by multiple mechanisms in Drosophila.

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.

Computational modeling offers new insight into Drosophila germ granule development.

The packaging of specific mRNAs into ribonucleoprotein granules called germ granules is required for germline proliferation and maintenance. During Drosophila germ granule development, mRNAs such as nanos (nos) and polar granule component (pgc) localize to germ granules through a stochastic seeding and self-recruitment process that generates homotypic clusters: aggregates containing multiple copies of a specific transcript. Germ granules vary in mRNA composition with respect to the different transcripts that they contain and their quantity. However, what influences germ granule mRNA composition during development is unclear. To gain insight into how germ granule mRNA heterogeneity arises, we created a computational model that simulates granule development. Although the model includes known mechanisms that were converted into mathematical representations, additional unreported mechanisms proved to be essential for modeling germ granule formation. The model was validated by predicting defects caused by changes in mRNA and protein abundance. Broader application of the model was demonstrated by quantifying nos and pgc localization efficacies and the contribution that an element within the nos 3' untranslated region has on clustering. For the first time, a mathematical representation of Drosophila germ granule formation is described, offering quantitative insight into how mRNA compositions arise while providing a new tool for guiding future studies.