Rif1 phosphorylation site analysis in telomere length regulation and the response to damaged telomeres

Abstract

Telomeres, the ends of eukaryotic chromosomes, consist of repetitive DNA sequences and their bound proteins that protect the end from the DNA damage response. Short telomeres with fewer repeats are preferentially elongated by telomerase. Tel1, the yeast homolog of human ATM kinase, is preferentially recruited to short telomeres and Tel1 kinase activity is required for telomere elongation. Rif1, a telomere-binding protein, negatively regulates telomere length by forming a complex with two other telomere binding proteins, Rap1 and Rif2, to block telomerase recruitment. Rif1 has 14 SQ/TQ consensus phosphorylation sites for ATM kinases, including 6 in a SQ/TQ Cluster Domain (SCD) similar to other DNA damage response proteins. These 14 sites were analyzed as N-terminal, SCD and C-terminal domains. Mutating some sites to non-phosphorylatable residues increased telomere length in cells lacking Tel1 while a different set of phosphomimetic mutants increased telomere length in cells lacking Rif2, suggesting that Rif1 phosphorylation has both positive and negative effects on length regulation. While these mutations did not alter the sensitivity to DNA damaging agents, inducing telomere-specific damage by growing cells lacking YKU70 at high temperature revealed a role for the SCD. Mass spectrometry of Rif1 from wild type cells or those induced for telomere-specific DNA damage revealed increased phosphorylation in cells with telomere damage at an ATM consensus site in the SCD, S1351, and non-ATM sites S181 and S1637. A phosphomimetic rif1-S1351E mutation caused an increase in telomere length at synthetic telomeres but not natural telomeres. These results indicate that the Rif1 SCD can modulate Rif1 function. As all Rif1 orthologs have one or more SCD domains, these results for yeast Rif1 have implications for the regulation of Rif1 function in humans and other organisms.

Proteomic analysis of bone marrow-derived mesenchymal stem cell extracellular vesicles from healthy donors: implications for proliferation, angiogenesis, Wnt signaling, and the basement membrane

“…RIF1 (Fig. 4 C) is a telomere-associated protein that plays a role in double-strand DNA breaks and promotes non-homologous end joining-mediated repair [ 35 – 37 ]. Specific cyclins were conserved among the donors’ BM-MSC EVs.…”

Rif1 regulates telomere length through conserved HEAT repeats

“…Rif1 is a known substrate of Tel1 kinase ( 24–26 ), and genetic analysis indicates that TEL1 and RIF1 are in the same pathway of telomere length regulation ( 26 , 27 ). While mutation of a cluster of S/T-Q motifs at positions 1308–1569 to AQ in the full-length Rif1 also did not affect telomere length ( 26 ), a recent paper implicated six S/T-Q motifs in the N-terminus of Rif1 as possibly playing a role in telomere length regulation ( 25 ). We wanted to test the hypothesis that Tel1 is epistatic to Rif1 because Tel1 phosphorylates Rif1 at S/T-Q sites in the Rif1 177–996 construct.…”

Rif1 regulates telomere length through conserved HEAT repeats

“…Rif1 is a known substrate of Tel1 kinase (24–26), and genetic analysis indicates that TEL1 and RIF1 are in the same pathway of telomere length regulation (26,27). A recent paper implicated six S/T-Q motifs in the N-terminus of Rif1 as possibly playing a role in telomere length regulation (25). Four of these sites are in the Rif1 177-996 construct and are the only S/T-Q motifs in this region.…”