Computational modeling offers new insight into Drosophila germ granule development

Valentino, Michael; Ortega, Bianca M; Ulrich, Bianca; Doyle, Dominique A; Farnum, Edward D.; Joiner, David A; Gavis, Elizabeth R.; Niepielko, Matthew G.

doi:10.1016/j.bpj.2022.03.014

Cited by 11 publications

(77 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We next explored the mechanisms by which Me31B influences nos localization. Recently, it has been shown using computational modeling and experimental validation that the localization of nos is influenced by 1) the number of nos transcripts that are expressed, 2) the amount of Osk in the system, and 3) nos ’s clustering factor, a quantifiable effect that, in conjunction with nos transcript and Osk levels, regulates the number of transcripts that can accumulate within a homotypic cluster [56, 57]. To identify whether me31B DVLAAAA affects one or more of these mechanisms, we first conducted RT-PCR to measure the level of nos and osk RNAs in the system.…”

Section: Resultsmentioning

confidence: 99%

“…smFISH, image analysis, and computational modeling smFISH was carried out as previously described using published nos probe sequences [56,57,70]. In summary, ovaries were dissected from yeast-fed females in cold PBS in under 10 minutes, and tissues were fixed for 30 minutes in 4% paraformaldehyde and PBS solution.…”

Section: Rna Extraction Cdna Synthesis and Quantitative Rt-pcrmentioning

confidence: 99%

“…The total number of homotypic clusters identified is reported in the figure legends. For modeling experiments, we employed a previously published model that simulates the formation of germ granules, including nos homotypic clusters [57].…”

Section: Rna Extraction Cdna Synthesis and Quantitative Rt-pcrmentioning

confidence: 99%

“…Data analysis was conducted by using the CFX Manager Software (BioRad) and Microsoft Excel. smFISH, image analysis, and computational modeling smFISH was carried out as previously described using published nos probe sequences [56,57,70]. In summary, ovaries were dissected from yeast-fed females in cold PBS in under 10 minutes, and tissues were fixed for 30 minutes in 4% paraformaldehyde and PBS solution.…”

Section: Rna Extraction Cdna Synthesis and Quantitative Rt-pcrmentioning

confidence: 99%

“…The only modeling parameters that were adjusted were 1) carrying capacity, which is regulated by Osk levels, was set from 1 (wild type) to 0.56, and 2) the pool of nos transcript expression was set from 1 (wild type) to 0.61 to mimic the RT-PCR levels reported in our results section [57]. *Note that two unintended, missense mutations were detected in the me31B FDF strain when sequencing its me31B gene.…”

Section: Rna Extraction Cdna Synthesis and Quantitative Rt-pcrmentioning

confidence: 99%

See 4 more Smart Citations

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

Kara

McCambridge

Proffer

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Rna Extraction Cdna Synthesis and Quantitative Rt-pcrmentioning

confidence: 99%

Section: Rna Extraction Cdna Synthesis and Quantitative Rt-pcrmentioning

confidence: 99%

Section: Rna Extraction Cdna Synthesis and Quantitative Rt-pcrmentioning

confidence: 99%

Section: Rna Extraction Cdna Synthesis and Quantitative Rt-pcrmentioning

confidence: 99%

See 3 more Smart Citations

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

Kara

McCambridge

Proffer

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

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

et al. 2023

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Drosophila germ granules are assembled from protein components through different modes of competing interactions with the multi‐domain Tudor protein

Wahiduzzaman,

Tindell,

Alexander

et al. 2024

FEBS Letters

View full text Add to dashboard Cite

Membraneless organelles are RNA–protein assemblies which have been implicated in post‐transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain‐containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that in Drosophila, different germ granule proteins associate with the multi‐domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudor in vitro and, surprisingly, partition to distinct and poorly overlapping clusters in germ granules in vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule.

show abstract

Computational modeling offers new insight into Drosophila germ granule development

Cited by 11 publications

References 61 publications

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

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

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

Drosophila germ granules are assembled from protein components through different modes of competing interactions with the multi‐domain Tudor protein

Contact Info

Product

Resources

About