Project description:Seb1 promotes co-transcriptional 3â?? end processing events that influence polyadenylation site decision knockdown experiments of Seb1 in Pombe
Project description:Termination of RNAPII transcription is associated with RNA 3â end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in S. cerevisiae do not rely on RNA cleavage for termination, but instead terminate via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the S. pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination, but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3â end processing factors, is enriched at the 3â end of genes, and binds RNA motifs downstream of cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3â UTR length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3â end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 co-transcriptionally controls alternative polyadenylation. Two biological replicates of Seb1 and Control (parental strain) CRAC experiments
Project description:Termination of RNAPII transcription is associated with RNA 3’ end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in S. cerevisiae do not rely on RNA cleavage for termination, but instead terminate via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the S. pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination, but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3’ end processing factors, is enriched at the 3’ end of genes, and binds RNA motifs downstream of cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3’ UTR length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3’ end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 co-transcriptionally controls alternative polyadenylation.
Project description:Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. Our project is to show that the conserved RNA-binding Seb1 is a pausing-promoting factor for RNA polymerases I to control cotranscriptional RNA processing. Here, we did a proximity dependent biotinylation followed by mass spectrometry (PDB-MS) of the Seb1 protein in order to assess for physical interactions with the RNAPI transcription machinery. A mutant E. coli BirA enzyme is fused to the Seb1 protein. This mutant version of BirA uses biotin to catalyze the formation of biotinoyl-5′-AMP (bioAMP), thereby generating a ‘cloud’ of activated biotin molecules that can react with free primary amines (most often lysine residues) of neighboring proteins. This experiment will support the conclusion that Seb1 is located at the rDNA locus.
