Project description:Ribosome biogenesis is a multi-step process, during which mistakes could take place at any step of pre-rRNA processing, modification, and assembly of ribosomes. Misprocessed rRNAs are usually detected and degraded by surveillance machineries. Recently, we identified a class of antisense ribosomal siRNAs (risiRNAs) that downregulate pre-rRNAs through the nuclear RNAi pathway. To further understand the biological roles of risiRNA, we conducted both forward and reverse genetic screenings to search for more susi mutants. We isolated a number of genes that are broadly conserved from yeast to humans and are involved in pre-rRNA modification and processing. Among them, SUSI-2(ceRRP8) is homologous to human RRP8 and engages in m1A methylation of 26S rRNA. C27F2.4(ceBUD23) is a m7G methyltransferase of 18S rRNA. E02H1.1(ceDIMT1L) is a predicted m6(2)Am6(2)A methyltransferase of 18S rRNA . Mutation of these genes led to modification deficiency of rRNAs and elicited accumulation of risiRNAs, which further triggered the cytoplasm to nuclear and nucleolar translocation of an Argonaute protein NRDE-3. The processing deficiency of rRNAs resulted in the accumulation of risiRNAs as well. We isolated SUSI-3(RIOK-1) which is similar to human RIOK1 that cleaves 20S to 18S rRNA. We further utilized RNAi and CRISPR/Cas9 technologies to perform candidate-based reverse genetic screening and identified additional pre-rRNA processing factors that suppressed risiRNA production. Therefore, we concluded that erroneous rRNAs can trigger risiRNA generation and subsequently turn on the nuclear RNAi-mediated gene silencing pathway to inhibit pre-rRNA expression, which may provide a quality control mechanism to maintain the homeostasis of rRNAs.
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately. Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA. Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2. Examination of targets for pre-rRNA endonucleases in yeast cells.
Project description:Coverage of short-read RNA-seq is highly non-uniform across transcripts even in genes with only one expressed isoform, contrary to biological expectation. We investigate the impact of several library preparation factors on the non-uniformity of coverage. Specifically, a mouse liver sample is prepared under varying ribosomal depletion (PolyA / rRNA digestion / rRNA pull-down / NoSelection), PCR ramp rate, and fragment length along with one heart sample without selection.
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately. Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA. Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2.