[
International Worm Meeting,
2021]
MicroRNAs (miRNAs) are short, non-coding RNAs that together with their protein cofactors, bind to target messenger RNAs to promote mRNA decay and translational repression. miRNAs are highly conserved and are implicated in essentially all biological processes in plants and animals. In the absence of miRNAs, C. elegans arrest during embryogenesis. Even partial loss of miRNAs can lead to severe development defects. The biogenesis of miRNAs is similar from C. elegans to humans, although there are differences in how primary miRNA transcripts are distinguished from other RNAs in different species and even between different miRNA transcripts within the same species. To better understand how miRNAs are processed in C. elegans, we developed an mCherry-based sensor that is desilenced when primary miRNA processing is impaired. We then performed a forward genetic screen to identify mutations that desilence the sensor and which may therefore affect primary miRNA recognition or processing. We are now characterizing a line we identified in our screen that has a mutation in the WW domain of Pasha as we more broadly explore the role of Pasha in primary miRNA recognition and processing.
[
International Worm Meeting,
2021]
Piwi-interacting RNAs (piRNAs) are a largely germline-specific class of small RNAs found in animals. Although piRNAs are best known for silencing transposons, they regulate many different biological processes. Here we identify a role for piRNAs in preventing runaway amplification of small interfering RNAs (siRNAs) from certain genes, including ribosomal RNAs (rRNAs) and histone mRNAs. In C. elegans, rRNAs and some histone mRNAs are heavily targeted by piRNAs, which facilitates their entry into an endogenous RNA interference (RNAi) pathway involving a class of siRNAs called 22G-RNAs. Under normal conditions, rRNAs and histone mRNAs produce relatively low levels of 22G-RNAs. But if piRNAs are lost, 22G-RNA production is highly elevated. We show that 22G-RNAs produced downstream of piRNAs likely function in a feed-forward amplification circuit. Thus, our results suggest that piRNAs facilitate low-level 22G-RNA production while simultaneously obstructing the 22G-RNA machinery to prevent runaway amplification from certain RNAs. Histone mRNAs and rRNAs are unique from other cellular RNAs in lacking polyA tails, which may promote feed-forward amplification of 22G-RNAs. In support of this, we show that the subset of histone mRNAs that contain polyA tails are largely resistant to silencing in piRNA mutants. Despite hyperproduction of 22G-RNAs in piRNA mutants, the effects on histone and rRNA expression are modest and may have a negligible impact on germline development.
[
International C. elegans Meeting,
2001]
Cell division results in the precise halving of genetic material. In yeast, such partitioning requires the protease separin 1 . This protease acts at the metaphase to anaphase transition to degrade sister chromatid cohesion, which in turn permits chromosomes to segregate to opposite poles of a dividing cell . In addition to facilitating chromosome segregation, separin promotes spindle elongation in S. cerevisiae through the activity of a calcium-binding domain 2 . Separin activity also depends heavily on the anaphase inhibitor securin, which localizes separin to both the nucleus and spindle mid-zone, and inhibits its protease activity until anaphase. Here, we report a requirement for the C. elegans homologue of yeast separin during oocyte meiosis. RNAi of this homologue, which contains both the protease and calcium binding domains of S. cerevisiae separin, results in multi-nucleated one-cell embryos with multiple spindles. In these embryos, the chromosomes are disorganized and the embryos fail to produce polar bodies. Taken together, these results suggest a role for C.elegans separin in chromosome segregation and cytokinesis in the early embryo. Currently, we are investigating if C. elegans separin also plays a role in exit from M-phase of the cell cycle. Additionally, we hope to identify protein partners of C.elegans separin by using the protein as bait in a yeast 2-hybrid screen. This screen may prove the most effective way of identifying a C. elegans securin homologue, as all of the known securins from other organisms demonstrate no amino acid conservation. Also, to date, no separin interacting proteins other than securin have been identified. Results of the screen will be discussed at the meeting. 1.Uhlman, F., Wernic, D., Poupart, M., Koonin, E., and K. Nasmyth. 2000. Cleavage of Cohesin by the CD Clan Protease Separin Triggers Anaphase in Yeast. Cell 103:375-386. 2.Jensen, S., Segal, M., Clarke, D., and S. Reed. 2001. A Novel Role of the Budding Yeast Separin Esp1 in Anaphase Spindle Elongation: Evidence that Proper Spindle Associatioin of Esp1 is Regulated by Pds1. J.Cell Biol. 152:27-40.