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Mapping of Sen1 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

ABSTRACT: Using CRAC, we compared the transcriptomic occupancy of Sen1 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 affected the binding of Sen1 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE276524 | GEO | 2025/08/25

REPOSITORIES: GEO

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Mapping of Nab3 RNA-binding sites in Saccharomyces cerevisiae with abrogated binding of Nab3 to PIC2

Project description:Using CRAC, we compared the transcriptomic occupancy of Nab3 in a Saccharomyces cerevisiae BY4741 parental strain (PIC2-GFP) and two derived mutants lacking Nab3 RNA-binding sites in PIC2. The purpose of the experiment was two-fold: on the one hand, we aimed to verify that the mutations inserted in the Nab3 binding sequences of PIC2 had indeed abrogated binding of Nab3 to the PIC2 transcript; on the other hand, we wanted to check whether differential binding of Nab3 to PIC2 would affect how the protein bound other targets in the genome. Sequencing outputs were processed using the pyCRAC pipeline and peak calling was performed with DBPeaks, our newly developed package for identification and comparison of binding sites defined by RNA-binding footprinting techniques (e.g., CRAC, iCLIP, PAR-CLIP, etc.).

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High-resolution transcriptome-wide maps of Sen1 RNA-binding by CRAC

Project description:Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a risk that needs to be controlled to prevent the perturbation of gene expression. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy terminal domain (CTD) of RNA polymerase II and characterize structurally its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires the recognition of the Ser5-phosphorylated form of the CTD by the N-terminal domain of Sen1. Furthermore, we find that the N-terminal and the C-terminal domains of Sen1 can mediate intra-molecular interactions. Our results shed light onto the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

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