Takatsuka, Hisashi et al. (2021) International Worm Meeting "Combinatorial analysis of human PAF1 complex-interacting proteins using in silico phylogenetic profiling and RNAi knockdown screening"

Ito, Masahiro, Kubota, Yukihiko, Takatsuka, Hisashi

[International Worm Meeting, 2021]

RNA polymerase-IIassociated factor-1 complex (PAF1C) is a heptameric protein complex consisting of PAF1, CTR9, LEO1, CDC73, and RTF1. PAF1C regulates gene transcription by interacting with RNA polymerase-II. PAF1C is evolutionarily conserved within multicellular organisms, and has been shown to regulate various important processes, including cell proliferation, cell differentiation, and tumor suppression. To date, the functional role of PAF1C in germ cell development remains unexplored. We previously reported that RNAi knockdown of PAF1C components renders animals oogenesis defective, as are the rtfo-1(tm5670) and leo-1(gk1081) mutants (International C. elegans conference 2019). In this work, we attempted to identify the molecular network underlying PAF1C's role in oogenesis by comprehensively analyzing proteins that interact physically with each human PAF1C component in silico. Phylogenetic profiles were generated for 542 species for which the whole genome sequence is available. These profiles allowed identification of 458 proteins that directly interact with PAF1C components in humans based on the presence or absence of orthologs. Further clustering analysis revealed a gene cluster comprising 192 conserved proteins in animals , and Blastp search revealed C. eleganshomologues (170 of 192). Among these we selected 12 C. elegans homologues that displayed all three of the following features: proteins that share similar expression with PAF1C in the gonad (55 of 170), proteins that produced an embryonic lethal phenotype in previous RNAi screened data (32 of 55), and proteins that localize to the nucleus (12 of 32). To examine the role of these 12 proteins in germ cell development, we compared each proteins RNAi knockdown phenotype with that of leo-1, which encodes one component of PAF1C. We found that 3 ubiquitin-related genes exhibit an oocyte-defective phenotype, as seen in leo-1(RNAi) animals. These results imply ubiquitin-mediated regulation of gene expression may play a role in PAF1C- dependent regulation of oogenesis. We plan to assess functional relationships between PAF1C and these factors in C. elegans oogenesis. Based on the results of these studies, we expect to elucidate a mechanism of oogenesis regulation that will aid in understanding and treating PAF1C-related reproductive organ pathologies in multicellular organism.

Kubota, Yukihiko et al. (2021) International Worm Meeting "The PAF1 complex cell-autonomously regulates oogenesis in Caenorhabditis elegans"

Ito, Masahiro, Ota, Natsumi, Unno, Takuma, Takatsuka, Hisashi, Kubota, Yukihiko

[International Worm Meeting, 2021]

During animal development, spatiotemporal gene transcription regulation is essential to regulate cell behavior. To precisely regulate gene transcription, regulation of the targeting of RNA polymerase II (pol II) to specific genes is required. RNA polymerase II-associated factor 1 complex (PAF1C) is a protein complex that consists of PAF1, CDC73, CTR9, LEO1, and RTF1, and has been shown to be involved in regulating pol II-mediated transcription. Although, it has been shown to regulate a variety of biological processes including cell differentiation and tumor suppression, a precise role for PAF1C during germ line development has not been clarified. The C. elegans hermaphrodite gonad produces sperm during late larval stage, which is then reserved in spermatheca. Subsequently, the gonad produces oocytes during the adult stage. Finally, gametogenesis is achieved via self-fertilization. At present, the mechanism of oogenesis is not fully understood. In this work, we found that although penetrance of leo-1 (RNAi) is lower than that for knockdown of the other four PAF1C component genes, oogenesis was not occurring in RNAi fed day1 adult stage escapers. Similar results were observed in leo-1 (gk1081), rtfo-1 (tm5670), and pafo-1 (tm13347) deletion mutants. We next checked the average number of oocyte maturation marker-positive oocytes using the bkcSi11[oma-1::GFP] genomic transgene driven by its ~2.9 kbp 5' region and its ~2.9 kbp 3'region. We observed about five OMA-1::GFP-positive oocytes per posterior gonad in both wild-type and control (RNAi) animals. By contrast, the number of OMA-1::GFP-positive oocytes was decreased in leo-1 (RNAi), cdc-73 (RNAi), pafo-1 (RNAi), and pafo-1 (tm13347) animals. We also found that functional PAFO-1::mCherry, which is expressed from an integrated genomic transgene, tjIs280[pafo-1p::pafo-1::mCherry] rescued the oogenesis defective phenotype in the pafo-1(tm13347) mutant. Moreover, functional PAFO-1::mCherry expressed by a regulatory element of the germ cell-specific gene pie-1 significantly rescued the oogenesis-defective phenotype. Thus, germ cell expression of PAF1C might be essential for oogenesis. Finally, we found bkcSi11[oma-1::GFP] partially rescued the oogenesis defective phenotype. Taken together, these results suggest that PAF1C cell-autonomously regulates oogenesis by regulating oocyte maturation.