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Npl3 stabilizes R-loops at telomeres to prevent accelerated replicative senescence

ABSTRACT: Telomere shortening rates must be regulated to prevent premature replicative senescence. TERRA R-loops become stabilized at critically short telomeres to promote their elongation through homology-directed repair (HDR), thereby counteracting senescence onset. Using a non-bias proteomic approach to identify telomere binding factors, we identified Npl3, an RNA-binding protein previously implicated in multiple RNA biogenesis processes. Using Chromatin- and RNA immunoprecipitation, we demonstrate that Npl3 interacts with TERRA and telomeres. Furthermore, we show that Npl3 associates to telomeres in an R-loop dependent manner, as changes in R-loop levels, e.g. at short telomeres, modulate the recruitment of Npl3 to chromosome ends. Through a series of genetic and biochemical approaches we demonstrate that Npl3 binds to TERRA and stabilizes R-loops at short telomeres, which in turn promotes HDR and prevents premature replicative senescence onset. This may have implications for diseases associated with excessive telomere shortening.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: F Butter

LAB HEAD: Falk Butter

PROVIDER: PXD016772 | Pride | 2020-01-31

REPOSITORIES: Pride

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			Action	DRS
	20160607_QEP2_FBU_LP_188_tlc10_telo.raw	Raw
	20160607_QEP2_FBU_LP_189_tlc10_control.raw	Raw
	20160607_QEP2_FBU_LP_190_tlc10_telo.raw	Raw
	20160607_QEP2_FBU_LP_191_tlc10_control.raw	Raw
	20160607_QEP2_FBU_LP_192_tlc12_telo.raw	Raw

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Project description:Alternative Lengthening of Telomeres (ALT) cancer cells are a subset of cancers that depend on the homologous recombination mechanism to extend their telomere length independent of telomerase. ALT cells contain elevated levels of the telomeric-repeat containing long noncoding RNA (TERRA), which is an RNA transcribed by RNA polymerase II and can form RNA:DNA (R-loops) hybrids at telomeres. Lines of evidence have shown that the formation of R-loops at telomeres could be one of the mechanisms to trigger DNA repair to lengthen telomeres. We perform iDRiP-MS, a method to capture specific RNA interacting protein by UV light crosslinking using antisense probe capture (Chu et al., 2017a; Minajigi et al., 2015), to explore the TERRA interactomes in human ALT cancer cell. Our TERRA interactome data reveals that TERRA interacts with an extensive subset of DNA repair proteins in ALT cells including the endonuclease XPF, suggesting that TERRA R-loops activate DDR via XPF to promote homologous recombination and telomere replication to drive ALT.

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