Rio2p, an Evolutionarily Conserved, Low Abundant Protein Kinase Essential for Processing of 20 S Pre-rRNA in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae Rio2p (encoded by open reading frame Ynl207w) is an essential protein of unknown function that displays significant sequence similarity to Rio1p/Rrp10p. The latter was recently shown to be an evolutionarily conserved, predominantly cytoplasmic serine/threonine kinase whose presence is required for the final cleavage at site D that converts 20 S pre-rRNA into mature 18 S rRNA. A data base search identified homologs of Rio2p in a wide variety of eukaryotes and Archaea. Detailed sequence comparison and in vitro kinase assays using recombinant protein demonstrated that Rio2p defines a subfamily of protein kinases related to, but both structurally and functionally distinct from, the one defined by Rio1p. Failure to deplete Rio2p in cells containing a GAL-rio2 gene and direct analysis of Rio2p levels by Western blotting indicated the protein to be low abundant. Using a GAL-rio2 gene carrying a point mutation that reduces the kinase activity, we found that depletion of this mutant protein blocked production of 18 S rRNA due to inhibition of the cleavage of cytoplasmic 20 S pre-rRNA at site D. Production of the large subunit rRNAs was not affected. Thus, Rio2p is the second protein kinase that is essential for cleavage at site D and the first in which the processing defect can be linked to its enzymatic activity. Contrary to Rio1p/Rrp10p, however, Rio2p appears to be localized predominantly in the nucleus.Like their counterparts in other eukaryotes, Saccharomyces cerevisiae ribosomes contain four species of rRNA: 5 S, 5.8 S, 18 S, and 25 S rRNAs. The genes encoding these rRNAs are organized on the yeast genome in 150 -200 tandem repeats, each of which comprises two transcriptional units separated by non-transcribed spacers. One of these units consists of a 5 S rRNA gene, transcribed by RNA polymerase III. The other unit contains single genes for each of the mature 18 S, 5.8 S, and 25 S rRNAs that are separated by internal transcribed spacers 1 and 2, whereas external transcribed spacer regions are present at either end of the unit (see Fig. 1A). After transcription of this polycistronic unit by RNA polymerase I, the spacers are removed from the primary transcript via an ordered series of endo-and exonucleolytic cleavages (see Fig. 1B) (reviewed in Refs. 1 and 2). The first detectable precursor species is 35 S pre-rRNA, which results from a cleavage at site B 0 in the 3Ј-external transcribed spacer by the yeast RNase III homolog Rnt1p (3,4). Subsequent cleavage at sites A 0 and A 1 in the 5Ј-external transcribed spacer first gives rise to 33 S and then 32 S pre-rRNA. The latter is cleaved at site A 2 to produce separate 20 S and 27 S A 2 precursors for the small and large ribosomal subunit, respectively.The majority (90%) of the 27 S A 2 precursor molecules are cleaved endonucleolytically at site A 3 , followed by exonucleolytic trimming to B 1S . The remainder are processed endonucleolytically 1 at site B 1L . The resulting 27 S B S and 27 S B L precursors, whose 5Ј-ends are located 6 nucleotide...

Dominant Mutations in the Late 40S Biogenesis Factor Ltv1 Affect Cytoplasmic Maturation of the Small Ribosomal Subunit in Saccharomyces cerevisiae

“…A large number of factors are required for this step, most of which must be involved in regulating the cleavage as only two, Nob1 and Fap7, are actually implicated in the cleavage step itself. Depletion of Rio1, Rio2, Tsr1, or Tsr2 all lead to 20S accumulation (Gelperin et al 2001;Vanrobays et al 2001Vanrobays et al , 2003Geerlings et al 2003;Peng et al 2003;Leger-Silvestre et al 2004). In the case of Rio2 and Tsr1, depletion results in the cytoplasmic accumulation of 20S as measured by FISH (Vanrobays et al 2003;Leger-Silvestre et al 2004), indicating that yeast Rio2 and Tsr1 function not in pre-40S export but in the cytoplasmic maturation of the 20SrRNA.…”

Powering through ribosome assembly

“…A Rio2 mutant in which in vitro kinase activity is largely abolished can support growth, at least when overexpressed (Geerlings et al 2003). Even though there are conflicting data concerning the ability of very low levels of wild-type but not kinase-deficient Rio2 to support growth (Geerlings et al 2003;Vanrobays et al 2003), it is likely that Rio2 is essential, but its kinase activity is not. This would be consistent with an inhibitory/regulatory role for this activity rather than an essential role.…”

“…Although both bind to pre-ribosomal particles containing the 20S rRNA precursor (Geerlings et al 2003;Vanrobays et al 2003), only Rio2 is a stable component of these particles (Gavin et al 2002;Schafer et al 2003;Collins et al 2007). Rio1 is not recovered with pre-ribosomes obtained by affinity purification of TAP-tagged accessory factors, indicating a transient association with pre-ribosomes, as expected if Rio1 phosphorylates a pre-ribosomal component and then dissociates.…”

Phosphorylation of Right Open Reading Frame 2 (Rio2) Protein Kinase by Polo-like Kinase 1 Regulates Mitotic Progression

“…FLAG antibody (M2) was purchased from Sigma. Myc (9E10), Plk1 (F-8), cyclin B1 (GNS1), ␤-actin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), and Ser(P)-10-H3 (sc-8656-R) antibodies were purchased from Santa Cruz Biotechnology. Antiphosphoserine antibody (AB1603) was purchased from Millipore.…”

“…Rio proteins, Rio1 and Rio2, are conserved from archaea to human, whereas Rio3 subfamily presents only in multicellular eukaryotes (13,14). Both yeast Rio1 and Rio2 are necessary for processing of 20 S pre-rRNA to the 18 S rRNA in 40 S ribosomal subunit synthesis (15,16). Human Rio2 (hRio2) was also found to be involved in late 40 S ribosomal subunit maturation (17,18).…”