Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs

Related exosome complexes of 335 exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg 2 -labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 335 exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3 processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47- strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3-extended nuclear pre-mRNAs or the cytoplasmic 335 mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs. The eukaryotic 18S, 5.8S, and 25S rRNAs (yeast nomenclature is given) are generated from a single large RNA polymerase I transcript by a series of endonucleolytic and exonucleolytic RNA processing reactions (reviewed in reference 39). The earliest detectable transcript in yeast, the 35S precursor rRNA (pre-rRNA), also undergoes extensive posttranscriptional modification involving predominantly pseudouridine formation and ribosyl-2-O-methylation. These modifications are directed to specific nucleotides within the 7-kb-long 35S prerRNA via complementary base-pairing mechanisms involving 70 different small nucleolar RNAs (snoRNAs).