The DEAD-box RNA helicase-like Utp25 is an SSU processome component

Abstract

The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A₀, A₁, and A₂. Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperature-sensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function. A yeast two-hybrid screen of Utp25 against other SSU processome components revealed several interacting proteins, including Mpp10, Utp3, and Utp21, thereby identifying the first interactions among the different subcomplexes of the SSU processome. Furthermore, the DUF1253 domain is required and sufficient for the interaction of Utp25 with Utp3. Thus, Utp25 is a novel SSU processome component that, along with Utp3, forms the first identified interactions among the different SSU processome subcomplexes.

FIGURE 1.

The pre-rRNA processing pathway in yeast. Cleavage at sites A to E (indicated in uppercase letters under the pre-rRNA schematic) releases the mature 18S, 5.8S, and 25S rRNAs from the 35S pre-rRNA transcript. The SSU processome guides cleavage at sites A₀ (in the 5′-ETS), A₁ (mature 5′-end of the 18S), and A₂ (in ITS1), thereby liberating the 18S rRNA from the 35S pre-rRNA. The 23S pre-rRNA is produced by an alternative processing pathway through an initial cleavage of the 35S pre-rRNA at site A₃. The lowercase letters above the rRNA schematic denote the oligonucleotide hybridization probes used in Figure 3B.

FIGURE 2.

Alignment of Utp25 with other DEAD-box RNA helicases. (A) Alignment of the yeast Utp25 with known yeast DEAD-box RNA helicases. The DEAD-box RNA helicases are presented in increasing order of sequence similarity to Utp25, with Prp28 being the least similar and Mak5 being the most similar. Numbers delineate the regions of Utp25 shown in the alignment. Areas of low sequence similarity outside of the conserved sequence blocks were removed and are depicted as gaps. Motif consensus sequences are derived from Cordin et al. (2006), with uppercase letters denoting well-conserved residues and conservative substitutions depicted by lowercase letters—“a” represents a conserved aromatic amino acid (F, W, or Y), “c” a charged group (D, E, H, K, or R), “h” a hydrophobic amino acid (A, F, G, I, L, M, P, V, W, or Y), “l” an aliphatic residue (I, L, or V), “o” an alcohol (S or T), “u” is A or G, “+” a positively charged group (H, K, or R), and “x” any residue. (B) The sequence of motif Ia is phylogenetically conserved among all eukaryotic supergroups. The complete alignment of Utp25 homologs can be found as Supplemental Figure S1, along with the sequence accession numbers.

FIGURE 3.

Utp25 is required for growth and for 18S pre-rRNA processing. (A) Genetic depletion of Utp25 after 24 h of growth in nonpermissive glucose medium impairs cell growth. (Inset) Utp25 and Utp6 (Dragon et al. 2002) levels (*) are reduced after genetic depletion for 24 h. Total yeast extract, separated on the same SDS-PAGE gel, was analyzed by Western blotting with an anti-HA antibody. A 50-kDa nonspecific cross-reacting band is also seen (•). (B) Genetic depletion of Utp25 reveals an 18S rRNA maturation defect. The parental strain YPH499 along with GAL∷3HA-UTP6 (Dragon et al. 2002) are shown as controls. Oligonucleotide hybridization probes c, b + e, and a + y are depicted in Figure 1 as lowercase letters above the rRNA schematic.

FIGURE 4.

Utp25 is an SSU processome component. The 3xHA-tagged Utp25 coimmunoprecipitates Mpp10 (A) and the U3 snoRNA (B), both known protein and RNA components of the SSU processome. Anti-HA antibodies were used to immunoprecipitate 3xHA-tagged Utp25, and Utp6 and Utp21, both known SSU processome components (Dragon et al. 2002; Bernstein et al. 2004), and the untagged parental strain YPH499. For the Western analysis, total input (T; 5%), mock IP with uncoupled beads (B), and the immunoprecipitates (IP) were separated by SDS-PAGE and blotted with an anti-Mpp10 antibody. For the Northern analysis, RNA was extracted from the total input (10%) and the IPs (as per the Western analysis), resolved on a denaturing gel and hybridized with a probe complementary to the U3 snoRNA. (C) Utp25 coimmunoprecipitates t-Utp8, Utp18, and Rrp9, members of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. The immunoprecipitations with anti-HA antibodies and Western analysis with anti-Mpp10 antibody were done as described above. The membrane was subsequently stripped and re-probed with anti-TAP antibody.

FIGURE 5.

The conserved motif Ia is not required for Utp25 function. (A) Wild-type and mutant motif Ia sequences, in which the residues were all mutated to alanines, of the yeast Utp25. (B) The full-length wild-type (p415-GPD∷UTP25) and mutant (p415-GPD∷utp25-motif Ia mutant) yeast UTP25 sequences were cloned into p415-GPD, a low-copy yeast expression vector with a constitutive GPD promoter, and transformed into the parental GAL∷3HA-UTP25 strain. (C) Complementation by the plasmid-borne wild-type or mutant Utp25 was assayed by rescue of the growth defect after depletion of endogenous Utp25 by growth on nonpermissive media (SD-Leu) at 30, 35, and 17°C after 7 d of growth. The untransformed parental GAL∷3HA-UTP25 strain, along with growth of all transformed strains on permissive medium (SG/R-Leu) enabling expression of the endogenous chromosomal Utp25, is also shown. (D) Growth curve of cells expressing no (squares), wild-type (circles), or motif Ia mutant (triangles) Utp25 proteins from the p415-GPD plasmid. Cells were grown in SG/R-Leu to exponential phase and transferred to SD-Leu. Cells were maintained in mid-log phage by frequent dilution, and growth was monitored for 24 h at 30°C.

FIGURE 6.

Utp25 and Utp3 link the UtpB and Mpp10 subcomplexes. (A) Re-streaks of the bait (underlined) and prey (shown surrounding the plate) protein–protein interaction screen from the 11 × 11 yeast two-hybrid array, focusing on previously unknown interactions involving Utp25 and Utp3. Yeast cells were assayed for growth on selective medium lacking leucine, tryptophan, and histidine and containing 3-AT. (B) Tabulation of the novel positive (+) and negative (−) protein–protein interactions involving Utp25 and Utp3. Previously known and recapitulated protein–protein interactions within the UtpB and Mpp10 subcomplexes are not shown. (C) Diagram of the obtained protein–protein interactions. Previously known and recapitulated protein–protein interactions are shown as lines; arrows pointing from bait to prey denote novel interactions. Double-headed arrows indicate interactions that were recovered as both bait and prey.

FIGURE 7.

The helicase-like domain of Utp25 mediates protein–protein interactions with Utp3. The full-length and N- and C-terminal truncations of Utp25 were cloned as bait, and the full-length Utp3 was cloned as prey, transformed into yeast and screened by yeast two-hybrid in an all-against-all mating array. Yeast cells were assayed for growth on selective medium, as described in the legend to Figure 6. The locations of the C-terminal truncations of Utp25, along with the N-terminal truncation corresponding to the helicase-like DUF1253 motif, are shown. The C-terminal truncations lie outside of the predicted location of the degenerate helicase motifs.

FIGURE 8.

Genetic depletion of Utp25 or of Utp3 does not alter SSU processome assembly. Cells were genetically depleted of Utp25 (A) or of Utp3 (B), and the SSU processome was coimmunoprecipitated with anti-Mpp10 antibodies. The presence of TAP-tagged t-Utp8, Utp18, or Rrp9 was assessed by Western analysis with anti-TAP antibodies. Genetic depletion of Utp25 or of Utp3 was confirmed by re-probing the blots with anti-HA antibodies.