Project description:Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.
Project description:Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.
Project description:Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.
Project description:Despite significant progress in the mechanistic understanding of epigenetic reprogramming of cells, the basis of 'organ reprogramming' by (epi-)gene-environment interactions remained largely obscured. Here, we use the ether-induced haltere-to-wing transformations in Drosophila as a model for epigenetic “reprogramming� at the whole organism level. Our findings support a mechanistic chain of events explaining why and how brief embryonic exposure to ether leads to organ transformation manifested at the larval stage and on. We show that ether interferes with protein integrity in the egg leading to altered deployment of Hsp90 and widespread repression of Trithorax-mediated establishment of H3K4 tri-methylations throughout the genome. Despite this global suppression of H3K4me3, Ubx targets, and wing development genes preferentially retain higher levels of active chromatin marks. This preferential retention pre-disposes Ubx targets and wing genes for later up-regulation in the larval haltere disc, hence the wing-like outcome. Consistent with compromised protein integrity during the exposure, the penetrance of bithorax transformation increases by genetic or chemical reduction of Hsp90 function. Moreover, joint reduction in Hsp90 and trx gene dosage can cause bithorax transformations even without exposure to ether, supporting underlying epistasis between Hsp90 and trx loss-of-functions. These findings implicate environmental disruption of protein integrity at the onset of histone methylations with a modification of epigenetic memory. The emerging picture provides a unique example in which the alleviation of the Hsp90 ‘capacitor function’ by the environment leads to a morphogenetic shift towards an ancestral-like body plan. The morphogenetic impact of chaperone response during a major setup of epigenetic patterns may be a general scheme for organ reprogramming by environmental cues.
Project description:Translation termination is an essential cellular process that is also of therapeutic interest for diseases that manifest from premature stop codons. In eukaryotes, translation termination requires eRF1, which recognizes stop codons, catalyzes the release of nascent proteins fom ribosomes, and facilitates ribosome recycling. The small molecule SRI-41315 triggers eRF1 degradation and enhances translational readthrough of premature stop codons. SRI-41315 promotes retention of eRF1 on ribosomes and leads to a higher frequency of translation termination at near-cognate stop codons. In this study, the systematic effect of SRI-41315 on translation termination at cognate and near-cognate stop codons is evaluated using a rabbit reticulocyte lysate-based in vitro translation system with endogenous transcript as well as reporter transcripts containing defined near-cognate stop codon.
Project description:Co-transcriptional RNA processing and surveillance factors mediate heterochromatin formation in fission yeast. In addition to RNAi, RNA elimination machinery including MTREC (Mtl1-Red1 core) and the exosome are involved in facultative heterochromatin assembly, however, the exact mechanisms remain unclear. Here we show that RNA elimination factors cooperate with the conserved exoribonuclease Dhp1/Rat1/Xrn2, which couples pre-mRNA 3â-end processing to transcription termination, to promote premature termination and facultative heterochromatin formation at meiotic genes. Dhp1 also affects termination of transcripts at genes that are targets of RNAi-mediated heterochromatin assembly. Moreover, Dhp1 facilitates constitutive heterochromatin formation and silencing at centromeric and mating-type loci. Remarkably, we find that Dhp1 interacts with the Clr4/Suv39h methyltransferase complex and acts directly to nucleate heterochromatin. Our results uncover a novel role for 3â-end processing and termination machinery in gene silencing through premature termination and suggest that non-canonical termination by Dhp1 and RNA elimination factors is linked to heterochromatin assembly. These findings have important implications for understanding mechanisms of gene silencing in higher eukaryotes. Sequencing and analysis of small RNA in two S. pombe mutants