Functional diversification of yeast telomere associated protein, Rif1, in higher eukaryotes.

BACKGROUND: Telomeres are nucleoprotein complexes at the end of linear eukaryotic chromosomes which maintain the genome integrity by regulating telomere length, preventing recombination and end to end fusion events. Multiple proteins associate with telomeres and function in concert to carry out these functions. Rap1 interacting factor 1 (Rif1), was identified as a protein involved in telomere length regulation in yeast. Rif1 is conserved upto mammals but its function has diversified from telomere length regulation to maintenance of genome integrity. RESULTS: We have carried out detailed bioinformatic analyses and identified Rif1 homologues in 92 organisms from yeast to human. We identified Rif1 homologues in Drosophila melanogaster, even though fly telomeres are maintained by a telomerase independent pathway. Our analysis shows that Drosophila Rif1 (dRif1) sequence is phylogenetically closer to the one of vertebrates than yeast and has identified a few Rif1 specific motifs conserved through evolution. This includes a Rif1 family specific conserved region within the HEAT repeat domain and a motif involved in protein phosphatase1 docking. We show that dRif1 is nuclear localized with a prominent heterochromatin association and unlike human Rif1, it does not respond to DNA damage by localizing to damaged sites. To test the evolutionary conservation of dRif1 function, we expressed the dRif1 protein in yeast and HeLa cells. In yeast, dRif1 did not perturb yeast Rif1 (yRif1) functions; and in HeLa cells it did not colocalize with DNA damage foci. CONCLUSIONS: Telomeres are maintained by retrotransposons in all Drosophila species and consequently, telomerase and many of the telomere associated protein homologues are absent, including Rap1, which is the binding partner of Rif1. We found that a homologue of yRif1 protein is present in fly and dRif1 has evolutionarily conserved motifs. Functional studies show that dRif1 responds differently to DNA damage, implying that dRif1 may have a different function and this may be conserved in other organisms as well.

Drosophila Rif1 is an essential gene and controls late developmental events by direct interaction with PP1-87B.

Rif1, identified as a regulator of telomerase in yeast, is an evolutionarily conserved protein and functions in diverse processes including telomere length regulation, epigenetic gene regulation, anti-checkpoint activity, DNA repair and establishing timing of firing at replication origins. Previously we had identified that all Rif1 homologues have PP1 interacting SILK-RVxF motif. In the present study, we show that Drosophila Rif1 is essential for normal fly development and loss of dRif1 impairs temporal regulation of initiation of DNA replication. In multiple tissues dRif1 colocalizes with HP1, a protein known to orchestrate timing of replication in fly. dRif1 associates with chromosomes in a mitotic stage-dependent manner coinciding with dephosphorylation of histones. Ectopic expression of dRif1 causes enlarged larval imaginal discs and early pupal lethality which is completely reversed by co-expression of PP1 87B, the major protein phosphatase in Drosophila, indicating genetic and functional interaction. These findings suggest that dRif1 is an adaptor for PP1 and functions by recruiting PP1 to multiple sites on the chromosome.