C.Allmang and J.Kufel contributed equally to this workThe yeast nuclear exosome contains multiple 3Ј→5Ј exoribonucleases, raising the question of why so many activities are present in the complex. All components are required during the 3Ј processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p. During this processing three distinct steps can be resolved, and hand-over between different exonucleases appears to occur at least twice. 3Ј processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi-step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component. The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3Ј extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3Ј end. This cleavage is followed by 3Ј→5Ј processing of the pre-snRNA involving the exosome complex and Dob1p. The exosome, together with Rnt1p, also participates in the 3Ј processing of the U1 and U5 snRNAs. We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions.

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-2Ј-O-methylation. These modifications are directed to specific nucleotides within the ϳ7-kb-long 35S prerRNA via complementary base-pairing mechanisms involving ϳ70 different small nucleolar RNAs (snoRNAs).The snoRNAs can be divided into two major functional groups; the box C/D snoRNAs direct methylation of ribosyl-2Ј-hydroxyl groups, whereas the H/ACA snoRNAs direct the conversion of uridine to pseudouridine (for reviews, see references 4 and 21). Genes encoding snoRNAs have a varied organization, but in yeast and mammals all are transcribed by RNA polymerase II. Most yeast snoRNA genes are expressed as individual transcripts from their own promoters, whereas several are processed from common primary transcripts and a few are encoded within the introns of protein-coding genes. Gene clusters are the predominant organization of snoRNA genes in plants, whereas the majority of mammalian snoRNAs are intron encoded.The synthesis of the mature 3Ј ends of all characterized snoRNAs requires endonucleolytic cleavage of the transcript, followed by 3Ј35Ј exonucleolytic processing. Endonucleolytic cleavage is by Rnt1p, the yeast RNase III homologue, or by cleavage factor 1A (CF1A), which is also required for the 3Ј end processing of mRNA transcripts (15). Maturation of intron-encoded snoRNAs involves linearization of the excised intron lariat, either by the debranching enzyme Dbr1p or endonucleolytic cleavage, followed by exonucleolytic proces...

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The exosome is a conserved multi-subunit ribonuclease complex that functions in 3 0 end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10-subunit core complex of the exosome (Exo-10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo-10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N-terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N-terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C-terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6-Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.