Abstract
U3 snoRNA is transcribed from two intron-containing genes in yeast, snR17A and snR17B. Although the assembly of the U3 snoRNP has not been precisely determined, at least some of the core box C/D proteins are known to bind pre-U3 co-transcriptionally, thereby affecting splicing and 3'-end processing of this snoRNA. We identified the interaction between the box C/D assembly factor Nop17p and Cwc24p, a novel yeast RING finger protein that had been previously isolated in a complex with the splicing factor Cef1p. Here we show that, consistent with the protein interaction data, Cwc24p localizes to the cell nucleus, and its depletion leads to the accumulation of both U3 pre-snoRNAs. U3 snoRNA is involved in the early cleavages of 35 S pre-rRNA, and the defective splicing of pre-U3 detected in cells depleted of Cwc24p causes the accumulation of the 35 S precursor rRNA. These results led us to the conclusion that Cwc24p is involved in pre-U3 snoRNA splicing, indirectly affecting pre-rRNA processing.
Highlights
The snoRNPs involved in rRNA modification can be divided into two major groups based on conserved sequence elements in the snoRNAs and on their association with evolutionarily conserved core proteins (6 – 8)
U3 snoRNA has been reported to associate with about 30 proteins [3, 24], and the final assembly of this snoRNP seems to occur during 35 S pre-rRNA processing [25]
Since Cwc24p interacts with Nop17p, a factor that affects assembly of box C/D snoRNP and, pre-rRNA processing [39], we investigated the possible involvement of Cwc24p in this process. ⌬cwc24/GAL1::CWC24 cells and isogenic haploid wild type cells were incubated in glucose-containing medium for 30 h and subsequently subjected to RNA pulse-chase labeling experiments with [3H]uracil. rRNA analysis from these cells shows that the 35 S pre-rRNA processing is much slower in the strain ⌬cwc24/GAL1::CWC24 than in the control CWC24 strain, since the precursor rRNA is still visible after 30 min of chase in the conditional strain, whereas it is no longer visible after 3 min of chase in the wild type strain
Summary
DNA Manipulation and Plasmid Construction—Plasmids used in this study, described in Table 1, were constructed according to the cloning techniques described by Sambrook et al [43] and sequenced by the Big Dye method (PerkinElmer Life Sciences). DNA Manipulation and Plasmid Construction—Plasmids used in this study, described, were constructed according to the cloning techniques described by Sambrook et al [43] and sequenced by the Big Dye method (PerkinElmer Life Sciences). CWC24 gene, encoded by the YLR323C open reading frame, was PCR-amplified from S. cerevisiae genomic DNA using primers 5Ј-CAAAGCTCCAGGAATTCATG-3Ј and 5Ј-GTTGATATGGGATCCTCCAT-3Ј. For the two-hybrid assays, the PCR product was digested with EcoRI and BamHI and cloned into pBTM116 [40] and pGAD-C2 [41] digested with the same enzymes, generating pBTM-CWC24 and pGAD-CWC24 (which code for the fusions BD-Cwc24p and AD-Cwc24p, respectively, where BD refers to the LexA DNA binding domain and AD refers to the Gal4p transcription activation domain). Plasmid pACT-CWC24 was isolated from a cDNA library obtained from the American Type Culture Collection (ATCC 87002) and consists of a GAL4-Cwc24p-(6 –260) fusion
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