Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Other
Summary
The non-polyadenylated mRNAs of replication-dependent histones (RDHs) and small nuclear RNAs (snRNAs) are synthesized by RNA polymerase II at histone locus bodies (HLBs) and Cajal bodies (CBs), respectively. We previously showed that MED26-containing Mediator regulates the 3'-end processing of non-polyadenylated transcripts by recruiting Little Elongation Complex (LEC) to the genes. HLBs frequently associate with CBs, in which 3'-end processing factors for RDH genes are enriched; however, this association’s role has not been elucidated. Here, we show that disruption of the Mediator docking site for LEC by mutating the MED26-binding site of EAF1, a subunit of LEC, interferes with CBs’ association with HLBs, resulting in decreased inter-chromosome interaction between RDH gene clusters and increased aberrant unprocessed RDH gene transcripts. Our findings suggest a model in which LEC recruitment by Mediator plays a role in CBs’ association with HLBs to facilitate the 3'-end processing of RDH genes by supplying 3'-end processing factors.
Overall design
Experiment 1: RNA-seq of ribosome RNA-depleted libraries prepared from HEK293T WT and EAF1-mutant cells. Experiment 2: PRO-seq in HEK293T WT and EAF1-mutant cells using D. melanogastor S2 cells as spike-in control. Experiment 3: ELL ChIP-seq in HEK293T WT cells. Experiment 4: 4C-seq in HEK293T WT and EAF1-mutant cells. Experiment 5: Coilin antibody-based in situ biotinylation-seq in HEK293T WT and EAF1-mutant cells using D. melanogastor S2 cells as spike-in control. Experiment 6: PRO-seq in siRNA-transfected HEK293T cells using D. melanogastor S2 cells as spike-in control. Experiment 7: PRO-seq in HEK293T WT and MED26-mutant cells using D. melanogastor S2 cells as spike-in control. Experiment 8: PRO-seq in alpha-amanitin-treated HEK293T cells. The read counts were normalized with million rRNA reads.
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