Reed, K. J. | Search bioRxiv

bioRxiv2024

Regulation of Microprocessor assembly and localization via Pasha's WW domain in C. elegans

Primary microRNA (pri-miRNA) transcripts are processed by the Microprocessor, a protein complex that includes the ribonuclease Drosha and its RNA binding partner DGCR8/Pasha. We developed a live, whole animal, fluorescence-based sensor that reliably monitors pri-miRNA processing with high sensitivity in C. elegans. Through a forward genetic selection for alleles that desilence the sensor, we identified a mutation in the conserved G residue adjacent to the namesake W residue of Pashas WW domain. Using genome editing we also mutated the W residue and reveal that both the G and W residue are required for dimerization of Pasha and proper assembly of the Microprocessor. Surprisingly, we find that the WW domain also facilitates nuclear localization of Pasha, which in turn promotes nuclear import or retention of Drosha. Furthermore, depletion of Pasha or Drosha causes both components of the Microprocessor to mislocalize to the cytoplasm. Thus, Pasha and Drosha mutually regulate each others spatial expression in C. elegans.

molecular biology↗

bioRxiv2020

piRNAs prevent runaway amplification of siRNAs from ribosomal RNAs and histone mRNAs

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 Caenorhabditis 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.

genetics↗