Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

Abstract

The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding prerRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components. Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions. Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology. By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p). The genes encoding both proteins are essential in yeast. Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA. Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA. Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly. However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity. Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events.Eukaryotic ribosomes are large ribonucleoproteins (RNPs) composed of four rRNAs and dozens of ribosomal proteins. Three of the four RNA components are processed from a single polycistronic transcript, undergo extensive nucleotide modification, and assemble with the appropriate proteins in the cell nucleolus. In the yeast Saccharomyces cerevisiae, the small-subunit (SSU) rRNA (18S) and the large-subunit (LSU) rRNAs (5.8S and 25S) are processed from the nascent 35S transcript via a series of cleavage steps (Fig. 1A). Successive cleavages at the 5Ј end of the precursor rRNA yield the 33S and 32S transcripts, while subsequent cleavage at the A2 site separates the precursors of the SSU rRNA from those of the LSU rRNA. The 20S pre-rRNA is matured to 18S rRNA, while the LSU precursors are terminally processed to the 5.8S and 25S rRNAs.Several small nucleolar RNPs (snoRNPs) are required for the cleavage steps that generate the mature 18S rRNA (reviewed in reference 35). Of these, the most widely studied is the U3 snoRNP. In yeast, genetic depletion of the U3 snoRNA results in a deficiency in processing at the A0, A1, and A2 sites, causing a sharp decrease in 20S and 18S rRNA levels and an apparent increase in the presence of the 23S precursor and the 35S nascent transcript (Fig. 1B). A role for the U3 snoRNP in pre-rRNA cleavage events has also been observed in metazoans. Oligonucleotide-mediated depletion of the U3 snoRNA in vivo in Xenopus laevis oocytes an...

Bms1p, a G-domain-containing protein, associates with Rcl1p and is required for 18S rRNA biogenesis in yeast

“…Rcl1p was previously demonstrated to specifically associate with U3 snoRNP + To find out whether Bms1p interacts with snoRNAs, we carried out IP reactions using the strain BMS1-ProtA, which chromosomally expresses a Bms1p-ProtA fusion+ Control strains expressing other ProtA-tagged proteins: Nop1p, a protein associated with U3 and other C/D box snoRNAs, Lsm3p, a protein associated with the U6 spliceosomal RNA, and Rcl1p, were analyzed in parallel+ As shown in Figure 7, Bms1p-ProtA specifically precipitates low levels of U3 snoRNA but none of the other RNAs tested, representing members of either the C/D-or H/ACA-box snoRNA families (U14, snR190 and snR10, snR30, respectively)+ Similar results were obtained when IPs performed with the anti-HA antibody were carried out with extracts originating from strains expressing either Bms1p-HA or the HA-tagged Imp3p, another protein associating specifically with the U3 snoRNP (Lee & Baserga, 1999; data not shown)+ Interestingly, also in this case immunoprecipitation of U3 snoRNA with Bms1p-HA was much less effective than with Imp3p-HA+ The significance of the less efficient precipitation of U3 snoRNA with Bms1p than with Rcl1p is not understood at present+ Low recovery of U3 RNA may be related to the observed susceptibility of Bms1p to proteolytic degradation (see Fig+ 2C)+ Alternatively, it is possible that interaction of the 10S complex with the nascent ribosome is accompanied by a structural rearrangement resulting in weakening of the interaction between Bms1p and Rcl1p and/or U3 snoRNP (see Discussion)+ FIGURE 5. Depletion of Bms1p causes defects in rRNA processing at cleavage sites A 0 , A 1 , and A 2 + A: Structure of 35S pre-rRNA (upper portion) and a simplified scheme of pre-rRNA processing in yeast (lower portion)+ Processing sites and intermediates are indicated+ For more details, see Kressler et al+ (1999) and Venema and Tollervey (1999)+ B: Northern blotting and primer extension analysis of RNA isolated from WT and GAL::bms1 strains grown in YPGal (lanes 0) or YPD6% for 6, 12, 22, or 44 h+ The oligonucleotide probes, indicated on the scheme of the 35S pre-rRNA above the gels, were complementary to: 59ETS, upstream of A 0 (a); ITS1, upstream of A 2 (b); ITS1, between A 2 and A 3 (c); ITS1, downstream of A 3 (d); ITS2, upstream of C 2 (e); mature 25S rRNA (f ); and mature 18S rRNA (g)+ Positions of different processing intermediates and mature rRNAs are indicated.…”

“…In this work, we show that Bms1p is a protein partner of Rcl1p in the yeast S. cerevisiae+ Like Rcl1p, Bms1p is an essential, evolutionarily conserved, nucleolar protein required for pre-rRNA processing at sites A 0 , A 1 , and A 2 , and the formation of 40S ribosomal subunits+ Bms1p and Rcl1p are components of a complex sedimenting on glycerol gradients at around 10S, which appears to associate with the U3 snoRNP at the level of nascent ribosomes+ Bms1p contains an evolutionarily conserved G-protein-like domain, suggesting that the protein may play an important regulatory role in pre-rRNA processing in the nucleolus+ Several independent lines of evidence support the conclusion that Bms1p is a true partner of Rcl1p+ The two proteins interact with each other in the yeast twohybrid system and coprecipitate together in extracts prepared from a strain expressing ProtA-tagged Bms1p+ Moreover, depletion of either Bms1p or Rcl1p results in a similar, though not identical, processing defect+ Both proteins strongly affect cleavages at sites A 1 and A 2 + However, the effect on processing at site A 0 is more pronounced in the absence of Bms1p than Rcl1p+ Bms1p and Rcl1p are detected in complexes of similar sizes+ Most significantly, association of Rcl1p with the prominent 10S complex is strongly diminished upon depletion of Bms1p, as is its association with structures sedimenting at ;40S and 70-90S that most likely represent nascent ribosomes at different stages of maturation+ This effect is Bms1p-specific, as depletion of Imp3p, another protein required for pre-rRNA processing at sites A 0 -A 2 (Lee & Baserga, 1999), does not result in any major change of Rcl1p distribution+ Despite the fact that neither Rcl1p nor Bms1p cosediments on gradients with the U3 monoparticle, both proteins specifically immunoprecipitate the U3 snoRNA, though with very different efficiencies+ Most probably, Rcl1p and Bms1p associate with U3 at the level of nascent ribosomes ; this work)+ The observation that the association of Rcl1p with the 70-90S and 40S complexes depends on the availability of Bms1p strongly argues for the importance of the Bms1p-Rcl1p interaction for this association+ Are Rcl1p and Bms1p the only components of the 10S complex? We have carried out experiments with extracts prepared from yeast strains expressing either Rcl1p or Bms1p tagged with the calmodulin binding peptide and ProtA (TAP-tag; Rigaut et al+, 1999)+ In FIGURE 7.…”

“…Glycerol gradient analysis has previously shown that Rcl1p is present in several different structures sedimenting at approximately 10S, 40S, and 70-80S; a fraction of the protein was also found to sediment as a free form near top of the gradient + To analyze the distribution of Bms1p, an extract prepared from strain BMS1-HA was layered on glycerol gradients, which were centrifuged for either 315 min or 24 h+ Fractions were collected and analyzed by westerns, using a-HA and a-Rcl1p Abs+ As shown in Figure 6A We investigated whether the effect on Rcl1p distribution results specifically from the depletion of Bms1p and not a general inhibition of pre-rRNA processing+ For this purpose we used the GAL::imp3 strain, which conditionally expresses the protein Imp3p+ Like Rcl1p, Imp3p associates specifically with the U3 snoRNP, and its depletion affects pre-rRNA processing at steps identical to those inhibited in the absence of Rcl1p or Bms1p (Lee & Baserga, 1999)+ We found that under permissive conditions the distribution of Rcl1p in the GAL::imp3 strain is similar to that seen with the BMS-HA strain (compare Fig+ 6A, lower row, with Fig+ 6B, third row) or the WT strain (not shown)+ Significantly, depletion of Imp3p, following 8 h of growth under repressive conditions, had no significant effect on the level of the 10S complex, and only marginally affected the level and the distribution of larger complexes containing Rcl1p (Fig+ 6B, bottom row)+ We have verified that depletion of Imp3p in the GAL::imp3 strain grown in YPD6% for 8 h causes the same changes in the polysome profile, including overaccumulation of the free 60S subunits, as depletion of Bms1p (not shown)+ These results demonstrate that the effect of Bms1p depletion on the Rcl1p sedimentation profile is specific+ T. Wegierski et al…”

“…In eukaryotic cells, synthesis and processing of ribosomal RNAs (rRNAs) and assembly of ribosomes occur in the nucleolus and follow an unusually complex pathway+ The 18S, 5+8S, and 25-28S rRNAs are synthesized as a single precursor (pre-rRNA), which contains additional sequences that are discarded during RNA maturation+ The maturation process involves extensive modification of rRNA nucleotides, most of them guided by small nucleolar RNAs (snoRNAs), followed by multiple cleavage events resulting in the formation of different processing intermediates+ The substrate for rRNA processing is a large ribonucleoprotein complex containing a multitude of ribosomal proteins and accessory nucleolar trans-acting factors that associate with the nascent pre-rRNA (reviewed by Kressler et al+, 1999;Venema & Tollervey, 1999;Lewis & Tollervey, 2000)+ rRNA processing has been most extensively studied in the yeast Saccharomyces cerevisiae and many trans-acting factors, both proteins and ribonucleoproteins, required for the process have been characterized+ These include, in addition to guide snoRNAs, the ribonucleoprotein RNase MRP, the essential snoRNAs U3, U14, snR30 and snR10, and many proteins, which either act in association with snoRNAs or function independently+ Among the latter are putative ATPdependent RNA helicases, Dim1p methylase, and endoand exoribonucleases (Kressler et al+, 1999;Venema & Tollervey, 1999)+ Despite substantial progress in identification of the trans-acting factors required for pre-rRNA processing, their precise functions remain largely unknown+ The factor best characterized to date is the U3 snoRNP+ Based on results from yeast and vertebrate systems, it appears that U3 snoRNP plays a central role in the assembly of the machinery responsible for processing of 18S rRNA and biogenesis of the 40S ribosomal subunit (Beltrame & Tollervey, 1992;Mougey et al+, 1993;Venema & Tollervey, 1999;Borovjagin & Gerbi, 2000, and references therein)+ U3 snoRNA base pairs with the 35S pre-rRNA within the 59 external transcribed spacer (ETS) and the 59 part of 18S rRNA and is required for early cleavages at the processing sites A 0 , A 1 , and A 2 (Beltrame & Tollervey, 1995;Sharma & Tollervey, 1999;Venema & Tollervey, 1999, and references therein)+ The yeast U3 snoRNP has recently been shown to contain five strongly associated structural proteins (Watkins et al+, 2000)+ However, consistent with the central role of U3 snoRNP in rRNA maturation, immunoprecipitation (IP) experiments revealed many additional proteins associating with the particle+ These include Sof1p, Mpp10p, Imp3p, Imp4p, Dhr1p, Lcp5p, and Rcl1p (Jansen et al+, 1993;Dunbar et al+, 1997;Wiederkehr et al+, 1998;Lee & Baserga, 1999;…”

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;…”