FBS Colloquia No.314Germline Biology Group
| Seminar or Lecture |
1. HemK2 is a methyltransferase ensuring the efficient protein production and mRNA stability during oogenesis in Drosophila melanogaster. Fengmei, Xu [D5, Kai Lab] 2. BioID-based biochemical screening and functional analysis of novel factors involved in piRNA-mediated transposon regulation in Drosophila melanogaster Wakana ISSHIKI [D2, Kai Lab] |
|---|---|
| Date and Time | 17 Nov. 2022 (Thu), 12:15～13:00 |
| Place | 2F Seminar Room, BioSystems Building and Online (Zoom) |
| Language | English |
| Contact |
Shinichi Kawaguchi |
1. HemK2 is a methyltransferase ensuring the efficient protein production and mRNA stability during oogenesis in Drosophila melanogaster.
HemK2 is a well-conserved methyltransferase from yeast to human, and three potential substrates, DNA N6-adenine (6mA), histone H4 and translation termination factor (eRF1), have been reported. Yet genuine substrates remain controversial. In this talk, I report that HemK2 functions for eRF1 methylation, which is required for female germline development in Drosophila melanogaster. Germline-knockdown of hemk2 (hemk2-GLKD) exhibited female sterility marked by developmental arrest in the mid-oogenesis; DNA double-strand break (DSB), upregulation of P53 gene, and massive apoptosis were observed. Upon hemk2-GLKD, eRF1 methylation at a conserved glutamine residue was significantly reduced. HPG (L-homopropargylglycine) protein synthesis assay revealed that hemk2-GLKD caused massive reduction of translational efficiency, possibly by perturbation of releasing efficient peptide at the stop codons. Intriguingly, defects during oogenesis in hemk2-GLKD were rescued by blocking of the no-go decay (NGD) pathway, suggesting that some mRNA were unstable under aberrant translational termination. Taken together, our results suggest that the eRF1 methylation by HemK2 is required for both the efficient protein production and mRNA stability, ensuring the proper progression of Drosophila oogenesis.
2. BioID-based biochemical screening and functional analysis of novel factors involved in piRNA-mediated transposon regulation in Drosophila melanogaster
As key defense mechanisms against transposons, PIWI-interacting (pi)RNA pathways play fundamental roles in maintaining genomic integrity in animal gonads. piRNAs form complexes with PIWI-clade Argonaute (Ago) family proteins and direct the silencing of transposons in post-transcriptional and transcriptional manners. Hence, PIWI proteins are central in piRNA-mediated transposon silencing. piRNA pathways involve a variety of factors including Tudor domain containing (TDRD) proteins and RNA helicases, which often interact with PIWI proteins. However, the molecular underpinnings of piRNA pathways have been elusive. Here, we developed a proteome screening system based at a proximity-dependent biotin identification (BioID/TurboID) to identify hitherto uncharacterized piRNA pathway factors in Drosophila melanogaster. In our system, an engineered biotin ligase, namely mini(m)Turbo, was fused to PIWI-clade members including Piwi, Aubergine (Aub), and Ago3. TurboID is inducible in undifferentiated ovarian germ cells, and biotinylated proteins were identified by mass spectrometry. The screening identified piRNA pathway factors in PIWI-selective manners, which ensure the specificity and sensitivity of our approach. We are performing systemic knock-down and knock-out approaches to other uncharacterized factors and would like to discuss about the results.
