Abstract
Xrn1p of Saccharomyces cerevisiae is a major cytoplasmic RNA turnover exonuclease which is evolutionarily conserved from yeasts to mammals. Deletion of the XRN1 gene causes pleiotropic phenotypes, which have been interpreted as indirect consequences of the RNA turnover defect. By sequence comparisons, we have identified three loosely defined, common 5′-3′ exonuclease motifs. The significance of motif II has been confirmed by mutant analysis with Xrn1p. The amino acid changes D206A and D208A abolish singly or in combination the exonuclease activity in vivo. These mutations show separation of function. They cause identical phenotypes to that of xrn1Δ in vegetative cells but do not exhibit the severe meiotic arrest and the spore lethality phenotype typical for the deletion. In addition, xrn1-D208A does not cause the severe reduction in meiotic popout recombination in a double mutant with dmc1 as does xrn1Δ. Biochemical analysis of the DNA binding, exonuclease, and homologous pairing activity of purified mutant enzyme demonstrated the specific loss of exonuclease activity. However, the mutant enzyme is competent to promote in vitro assembly of tubulin into microtubules. These results define a separable and specific function of Xrn1p in meiosis which appears unrelated to its RNA turnover function in vegetative cells.
ACKNOWLEDGMENTS
We thank D. Botstein, N. Kleckner, and R. Kolodner for kindly supplying strains. The pRDK249 overexpression vector was kindly provided by A. Johnson and R. Kolodner. The plasmid containing the mouse β-actin gene for transcription was kindly supplied by T. Seebeck. We are grateful to S. Edelstein for support and helpful discussion and to I. Andrey Tornare for skillful technical assistance. We thank all members of the Heyer laboratory, especially V. Bashkirov, for help and useful discussions.
This work was supported by a career development award (START) to W.D.H., research grants of the Swiss National Science Foundation to W.D.H. and S. Edelstein, and funds from UC Davis to W.D.H.