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

Abstract

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...

Inactivation of Cleavage Factor I Components Rna14p and Rna15p Induces Sequestration of Small Nucleolar Ribonucleoproteins at Discrete Sites in the Nucleus

“…Total poly(A) ϩ RNA staining was also greatly reduced in the rna14-1 and rna15-2 strains incubated at 37°C for 1 h (Figure 4, h-j), consistent with previous reports (Minvielle-Sebastia et al, 1991;Torchet et al, 2002). By contrast, in strains lacking only Rrp6p (Figure 4, c-e), a strong accumulation of polyadenylated RNA was observed in the nucleus, as reported previously (Allmang et al, 1999a;van Hoof et al, 2000;Mitchell et al, 2003;Fang et al, 2004;Kuai et al, 2004;Carneiro et al, 2007). To allow the direct comparison of fluorescence intensities, images from the rrp6-⌬ mutant incubated at 23°C ( Figure 4C) and 37°C ( Figure 4D) were acquired using the same microscope settings.…”

“…Yeast RNA extraction, Northern blotting, and deadenylation analysis were performed as described previously (Mitchell et al, 2003;LaCava et al, 2005).…”

The PNPase, exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

“…(41/42-like heterodimer) [31] Cofactors for the exosomes without 3¢ exoRNase activity RhlB RNA helicase [50] Degradosome complex in certain bacteria [51] Ski7p and Ski RNA helicase complex [52,53] Mtr4p RNA helicase and TRAMP complex [54], Rrp47 [48], Nrd1 [55] RNA helicase associated with AU-rich element or RHAU [56], KSRP [57], TTP [58] a It is not known yet whether human nuclear and cytoplasmic exosomes contain different components.…”

Exosome Cofactors Connect Transcription Termination to RNA Processing by Guiding Terminated Transcripts to the Appropriate Exonuclease within the Nuclear Exosome

“…Recently, Trf4 was shown to interact with the Nrd1 CID, suggesting that degradation of Nrd1-terminated transcripts by the exosome is coordinated at least in part via Trf4 (19). Other known cofactors of the nuclear exosome are Rrp47 (C1D in humans) and Mpp6 (20,21), which preferentially bind to structured and pyrimidine-rich RNAs, respectively (21,22). Rrp47 directly interacts with the PMC2NT domain of Rrp6 (22) and forms a composite surface for recruiting Mtr4 (23), suggesting that Rrp47 and TRAMP may be functionally linked to the activity of Rrp6.…”