Multiple genetic and biochemical interactions of Brr2, Prp8, Prp31, Prp1 and Prp4 kinase suggest a function in the control of the activation of spliceosomes in Schizosaccharomyces pombe
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The spliceosomal component Prp1 (U5-102 kD) is found in Schizosaccharomyces pombe, a physiological substrate of Prp4 kinase. Here, we identify, spp41-1, a previously isolated extragenic suppressor of Prp4 kinase. The gene encodes an ATP-dependent RNA helicase homologous to the splicing factor Brr2 of Saccharomyces cerevisiae and U5-200 kD of mammalia. The suppressor allele, spp41-1, interacts genetically with alleles of prp1. We show that Prp1 and Brr2 are complexed in vivo with spliceosomal particles containing the five snRNAs U1, U2, U5, and base-paired U4/U6. Prp1 was found exclusively in small ribonucleoprotein particle (snRNP) complexes sedimenting in the range of 30S–60S, whereas Brr2 was also found sedimenting lower than 30S and free of snRNAs. Moreover, we find that the splicing factor Prp31 is complexed with Prp1 in the same spliceosomal particles containing the five snRNAs. These data indicate that in fission yeast spliceosomal particles larger than 30S exist, which can be considered as pre-catalytic spliceosomes. In addition, we show that S. pombe cells lacking Prp1 still contain these large pre-catalytic spliceosomal particles associated with Prp31. These data are consistent with the notion that in fission yeast phosphorylation of Prp1 by Prp4 kinase is involved in the activation of pre-catalytic spliceosomes.
KeywordsFission yeast Pre-mRNA splicing Spliceosomal complexes Prp4 kinase Prp1 Brr2 Prp8
We thank Susanne Zock-Emmenthal for her excellent technical assistance. We are grateful to Ralf Schnabel (Technical University of Braunschweig, Germany) for stimulating and fruitful discussions and valuable comments on the manuscript. We thank Reinhard Lührmann (MPI, Göttingen, Germany) for the TMG antibodies, Chris Norbury (University of Oxford, UK) for the Cdc2 antibodies, Kathleen Gould (Vanderbilt School of Medicine, USA) for a HACdc5 tagged strain, and Knud Nierhaus (MPI, Berlin, Germany) for E. coli ribosomal subunits. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to NFK.
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