ASH1 mRNA localizes to the bud tip in Saccharomyces cerevisiae to establish asymmetry of HO expression, important for mating type switching. To visualize real time localization of the mRNA in living yeast cells, green fluorescent protein (GFP) was fused to the RNA-binding protein MS2 to follow a reporter mRNA containing MS2-binding sites. Formation and localization of a GFP particle in the bud required ASH1 3'UTR (untranslated region) sequences. The SHE mutants disrupt RNA and particle localization and SHE 2 and 3 mutants inhibit particle formation as well. Both She3myc and She1myc colocalized with the particle. Video microscopy demonstrated that She1p/Myo4p moved particles to the bud tip at 200-440 nm/sec. Therefore, the ASH1 3'UTR-dependent particle serves as a marker for RNA transport and localization.

Dnmt2 proteins are the most conserved members of the DNA methyltransferase enzyme family, but their substrate specificity and biological functions have been a subject of controversy. We show here that, in addition to tRNA Asp-GTC , tRNA Val-AAC and tRNA Gly-GCC are also methylated by Dnmt2. Drosophila Dnmt2 mutants showed reduced viability under stress conditions, and Dnmt2 relocalized to stress granules following heat shock. Strikingly, stress-induced cleavage of tRNAs was Dnmt2-dependent, and Dnmt2-mediated methylation protected tRNAs against ribonuclease cleavage. These results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for Dnmt2 enzymes during the biogenesis of tRNA-derived small RNAs.Supplemental material is available at http://www.genesdev.org.

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerasedependent telomere elongation is blocked by sequestration of the 3 telomere terminus in TRF1-and TRF2-induced telomeric loops.The length of mammalian telomeres is governed by a homeostasis mechanism. Telomeres have a species-specific length setting (26) which is constant over the generations, despite high levels of telomerase activity in the germline (47). For instance, the telomeres of Mus musculus are maintained at 20 to 50 kb, while the closely related mouse species Mus spretus has telomeric tracts closer in length to human telomeres, usually ranging from 5 to 15 kb. Similarly, despite high levels of telomerase, the telomeres of many human tumor cell lines do not grow but are stably maintained at a setting characteristic for each individual cell line (12,13,44). Telomere length homeostasis is also evident when new telomeres are generated by transfection of short stretches of telomeric DNA into cultured cells. The transfected telomeric tracts are elongated, presumably by telomerase, until their length matches the other telomeres in the transfected cells (3,23,43). The new telomere presumably recruits telomere binding proteins and so attains all functions of telomeres, including the length regulation characteristic of a given cell. Regulated growth of a single telomere, as observed in this context, suggests that cells can measure and modulate the length of the telomeric repeat array at individual chromosome ends, implying a cis-acting regulatory mechanism.Human telomeres contain two related TTAGGG repeat binding factors, TRF1 and TRF2 (7, 10, 11). Both TRF proteins have a Myb-like helix-turn-helix domain in their carboxy terminus and a central conserved domain that includes sequences responsible for the formation of homodimers. The two proteins do not heterodimerize, and ...

The function of cytosine-C5 methylation, a widespread modification of tRNAs, has remained obscure, particularly in mammals. We have now developed a mouse strain defective in cytosine-C5 tRNA methylation, by disrupting both the Dnmt2 and the NSun2 tRNA methyltransferases. Although the lack of either enzyme alone has no detectable effects on mouse viability, double mutants showed a synthetic lethal interaction, with an underdeveloped phenotype and impaired cellular differentiation. tRNA methylation analysis of the double-knockout mice demonstrated complementary target-site specificities for Dnmt2 and NSun2 and a complete loss of cytosine-C5 tRNA methylation. Steady-state levels of unmethylated tRNAs were substantially reduced, and loss of Dnmt2 and NSun2 was further associated with reduced rates of overall protein synthesis. These results establish a biologically important function for cytosine-C5 tRNA methylation in mammals and suggest that this modification promotes mouse development by supporting protein synthesis.