Nop58p is a common component of the box C+D snoRNPs that is required for snoRNA stability

Abstract

Eukaryotic nucleoli contain a large family of box C+D small nucleolar RNA (snoRNA) species, all of which are associated with a common protein Nop1p/fibrillarin. Nop58p was identified in a screen for synthetic lethality with Nop1p and shown to be an essential nucleolar protein. Here we report that a Protein A-tagged version of Nop58p coprecipitates all tested box C+D snoRNAs and that genetic depletion of Nop58p leads to the loss of all tested box C+D snoRNAs. The box H+ACA class of snoRNAs are not coprecipitated with Nop58p, and are not codepleted. The yeast box C+D snoRNAs include two species, U3 and U14, that are required for the early cleavages in pre-rRNA processing. Consistent with this, Nop58p depletion leads to a strong inhibition of pre-rRNA processing and 18S rRNA synthesis. Unexpectedly, depletion of Nop58p leads to the accumulation of 39 extended forms of U3 and U24, showing that the protein is also involved in snoRNA synthesis. Nop58p is the second common component of the box C+D snoRNPs to be identified and the first to be shown to be required for the stability and for the synthesis of these snoRNAs.

An unexpected, conserved element of the U3 snoRNA is required for Mpp10p association

“…The hinge region, the spacing between the 59 and 39 domains of the U3 snoRNA, is one of the least understood segments of the U3 snoRNA+ In yeast, the hinge region encompasses nt 39 to 72+ A hairpin called helix 1b9 has been proposed to exist in snoRNPs in solution within these boundaries (Segault et al+, 1992;Mereau et al+, 1997)+ Helix 1b9 is sometimes drawn as part of a larger helix termed helix 1b or stem-loop 1b (Segault et al+, 1992;Mereau et al+, 1997;Antal et al+, 2000)+ Although the primary sequence of the hinge region differs among eukaryotic organisms, its length is fairly well conserved (between 34 and 37 nt; Samarsky & Fournier, 1998)+ The 59 portion of the hinge region (nt 39-48) base pairs with the pre-rRNA as a prerequisite for the cleavage reaction at A0 (Beltrame & Tollervey, 1992, 1995Beltrame et al+, 1994)+ Therefore portions of both the 59 domain and the hinge region base pair with the pre-rRNA to effect pre-rRNA cleavage+ Because of these RNA-RNA interactions, the two hairpins in the 59 domain and hinge region of the U3 snoRNA are not likely to exist in vivo (Antal et al+, 2000;Borovjagin & Gerbi, 2000)+ The U3 snoRNA is complexed with both proteins common to box C/D snoRNPs and proteins specific for the U3 snoRNP+ The common proteins, conserved from yeast to humans, are fibrillarin (Nop1p), Nop56p, Nop5/ Nop58p, and Snu13p (Schimmang et al+, 1989;Henriquez et al+, 1990;Tollervey et al+, 1991Tollervey et al+, , 1993Gautier et al+, 1997;Wu et al+, 1998;Lafontaine & Tollervey, 1999;Watkins et al+, 2000)+ In addition, the U3 snoRNP contains at least seven other proteins that are not components of any other snoRNP-Sof1p, Mpp10p, Imp3p, Imp4p, Lcp5p, Rrp9p, and Dhr1p (Jansen et al+, 1993;Dunbar et al+, 1997;Wiederkehr et al+, 1998;Wu et al+, 1998;Lee & Baserga, 1999;Colley et al+, 2000;Venema et al+, 2000)+ Mpp10p, Imp3p, Imp4p, Rrp9p, and the common proteins have known metazoan homologs (Gautier et al+, 1997;Pluk et al+, 1998;…”

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

“…Structure of the pre-rRNA and processing pathway in Saccharomyces cerevisiae. A: Structure of the 35S pre-rRNA+ The 35S operon contains the sequences for the mature 18S, 5+8S, and 25S rRNAs separated by the two internal transcribed spacers (ITS1 and ITS2)+ In addition, two external transcribed spacers, the 59ETS and 39ETS, are present at either end+ B: Pre-rRNA processing pathway+ The 35S pre-rRNA undergoes sequential processing reactions to generate the mature rRNAs+ Cleavage at A0 generates the 33S pre-rRNA+ It is subsequently cleaved at sites A1 at the 59 end of the 18S rRNA giving rise to the 32S pre-rRNA, and A2 in ITS1 yielding the 20S and 27SA2 pre-rRNAs+ The latter two species are precursors for the rRNAs respectively found in the small and large ribosomal subunits+ The 20S pre-rRNA is endonucleolytically cleaved at site D to produce the 18S rRNA+ The 27SA2 intermediate is processed by two alternative pathways+ In the major pathway, 27SA2 is cleaved by RNase MRP at site A3 in ITS1 yielding 27SA3, which is trimmed by 5939 exonucleases Rat1p and Xrn1p to site B1 S , generating the 27SB S precursor+ Approximately 15% of the 27SA2 molecules are processed at site B1 L , producing the 27SB L pre-rRNA+ 27SB S and 27SB L molecules are then processed at sites C1 and C2, yielding mature 25S rRNA and the 7S S and 7S L pre-rRNAs respectively, which are converted to 5+8S S and 5+8S L rRNAs by a complex of 39 59 exoribonucleases, the "exosome+" shifted to their associated proteins+ C/D snoRNPs are now known to contain three common essential proteins, Nop1p/fibrillarin (Tyc & Steitz, 1989, Baserga et al+, 1991Peculis & Steitz, 1994;Ganot et al+, 1997b), Nop56p, and Nop58p/Nop5p (Gautier et al+, 1997;Wu et al+, 1998, Lafontaine & Tollervey, 1999, whereas H/ACA snoRNPs contain at least four, Cbf5p (Jiang et al+, 1993;Cadwell et al+, 1997;, Gar1p (Girard et al+, 1992, Ganot et al+, 1997bHenras et al+, 1998), Nhp2p (Henras et al+, 1998;Watkins et al+, 1998), and Nop10p (Henras et al+, 1998)+ The discovery that Cbf5p, which is highly related to pseudouridine synthases (Koonin, 1996), is an integral snoRNP component strongly suggests that H/ACA snoRNPs not only select, but also modify prerRNA uridine residues )+ C/D snoRNPs also probably catalyze pre-rRNA modifications, as it has recently been noticed that the first characterized snoRNP protein component, Nop1p/ fibrillarin, contains the conserved methyltransferase motifs (Niewmierzycka & Clarke, 1999)+ SnoRNPs need to be able to reversibly interact with the pre-rRNA to catalyze pre-rRNA modifications or to promote pre-rRNA cleavage events+ Little is known about the ways snoRNPs/pre-rRNA interactions are modulated+ In the absence of the Gar1p protein, so-called because it contains two glycine/arginine-rich (GAR) domains at its N-and C-ter...…”

Interaction of the U3-55k protein with U3 snoRNA is mediated by the Box B/C motif of U3 and the WD repeats of U3-55k

“…The RNA is associated with proteins common to all Box C/D snoRNAs, including fibrillarin/Nop1p (34,35), Nop56p (36) and Nop58/Nop5p (37)(38)(39) and proteins specific to the U3 snoRNP, including Sof1p (supressor of fibrillarin; 40), Mpp10 (41,42), Lcp5p (43), Imp3p (44), Imp4p (44) and U3-55k (29,45). Each of these proteins (like U3 RNA) are essential and have been shown to be specifically required for the maturation of 18S rRNA [39][40][41]43,44,46,47 personal communication].…”