Pac1p, an RNase III homolog, is required for formation of the 3 ' end of U2 snRNA in Schizosaccharomyces pombe

Like its homologs in higher eukaryotes, the U2 snRNA in Schizosaccharomyces pombe is transcribed by RNA polymerase Ii and is not polyadenylated. Inste ad, an RNA stem-loop structure located downstream of the U2 snRNA coding se quence and transcribed as part of a 3' extended precursor serves as a signa l for 3'-end formation. We have identified three mutants that have temperat ure-sensitive defects in U2 snRNA 3'-end formation, In these mutants, the s ynthesis of the major snRNAs is also affected and unprocessed rRNA precurso rs accumulate at the restrictive temperature. Two of these mutants contain the same G-to-A transition within the pad gene, whereas the third contains a lesion outside the pad locus, indicating that at least two genes are invo lved, The pac1(+) gene is codominant with the mutant allele and can rescue the temperature-sensitive phenotype and the defects in snRNA and rRNA synth esis, if overexpressed. In vitro, Pac1p, an RNase III homolog, can cleave a synthetic U2 precursor within the signal for 3'-end formation, generating a product that is a few nucleotides longer than mature U2 snRNA, In additio n, U2 precursors are cleaved and trimmed to the mature size in extracts mad e from wild-type S. pombe cells. However, extracts made from pad mutant cel ls are unable to do so unless they are supplemented with purified recombina nt Pac1p, Thus, the 3' end of S, pombe U2 snRNA is generated by a processin g reaction that requires Pac1p and an additional component, and can be diss ociated from transcription in vitro.