Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE110028 | GEO | 2018/11/21
REPOSITORIES: GEO
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Project description:Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNA are generally short-lived, length-restricted and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly depletion of the ubiquitous Pol II associated transcription elongation factor SPT6, promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of Integrator complex to chromatin which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNA that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNA overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall our results underline the importance of restricting lncRNA expression.
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Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.
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Project description:RNA-mediated transcriptional silencing prevents deleterious effects of transposon activity and controls the expression of protein-coding genes. It involves long non-coding RNAs (lncRNAs)1, which in Arabidopsis thaliana are produced by a specialized RNA Polymerase V (Pol V)2. lncRNAs guide Argonaute-siRNA complexes to specific genomic loci and mediate the establishment of DNA methylation3,4. The mechanism by which lncRNAs affect chromatin structure and mRNA production remains mostly unknown. Here we identify the SWI/SNF nucleosome remodeling complex as a component of the RNA-mediated transcriptional silencing pathway. We found that SWI3, an essential subunit of the SWI/SNF complex, physically interacts with a lncRNA-binding IDN2 protein5,6. RNA-mediated DNA methylation and transcriptional silencing was compromised in the swi3 mutant. Moreover, targets of SWI/SNF significantly overlapped with genes controlled by Pol V, which shows that the physical interaction reflects a functional relationship. We further found that non-coding transcription by Pol V affects nucleosome positioning on silenced regions. We propose that lncRNAs mediate transcriptional silencing by guiding the SWI/SNF complex and establishing positioned nucleosomes on specific genomic loci. We further propose that guiding ATP-dependent chromatin remodeling complexes may be a more general function of lncRNAs. H3 ChIP-seq of 2 samples (Col-0 and nrpe1) with 2 biological repeats.
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