Inactivation of Cleavage Factor I Components Rna14p and Rna15p Induces Sequestration of Small Nucleolar Ribonucleoproteins at Discrete Sites in the Nucleus

Abstract

Small nucleolar RNAs (snoRNAs) associate with specific proteins forming small nucleolar ribonucleoprotein (snoRNP) particles, which are essential for ribosome biogenesis. The snoRNAs are transcribed, processed, and assembled in snoRNPs in the nucleoplasm. Mature particles are then transported to the nucleolus. In yeast, 3-end maturation of snoRNAs involves the activity of Rnt1p endonuclease and cleavage factor IA (CFIA). We report that after inhibition of CFIA components Rna14p and Rna15p, the snoRNP proteins Nop1p, Nop58p, and Gar1p delocalize from the nucleolus and accumulate in discrete nucleoplasmic foci. The U14 snoRNA, but not U3 snoRNA, similarly redistributes from the nucleolus to the nucleoplasmic foci. Simultaneous depletion of either Rna14p or Rna15p and the nuclear exosome component Rrp6p induces accumulation of poly(A)؉ RNA at the snoRNP-containing foci. We propose that the foci detected after CFIA inactivation correspond to quality control centers in the nucleoplasm. INTRODUCTIONSmall nucleolar RNAs (snoRNAs) are an abundant group of nonprotein coding RNAs in eukaryotic cells (Kiss, 2002). The snoRNAs associate with specific proteins forming small nucleolar ribonucleoprotein (snoRNP) particles, the majority of which participates in the biogenesis of ribosomes in the nucleolus (for recent reviews, see Bachellerie et al., 2002;Filipowicz and Pogacic, 2002;Kiss, 2002;Tran et al., 2004). The known snoRNAs fall into two major classes named box C/D and box H/ACA, which are characterized by distinctive sequence elements and associated proteins.The snoRNAs are transcribed as precursors that undergo posttranscriptional processing to generate the mature functional forms. Although most vertebrate snoRNAs are processed from the intronic regions of mRNA precursors Fournier, 1995, Bachellerie et al., 2002), only a few yeast snoRNAs are intron encoded. The majority of yeast snoRNAs are encoded in independent monocistronic or polycistronic transcripts that are transcribed by RNA polymerase II. Intronic snoRNAs can be produced either by splicing-dependent or by splicing-independent processing, whereas the maturation of independently transcribed units involves both endonucleolytic cleavage and exonucleolytic trimming (Filipowicz and Pogacic, 2002).In yeast, the U14 and U3 box C/D snoRNAs constitute paradigms for the study of snoRNA biosynthesis. U14 is cotranscribed with snR190, another box C/D snoRNA (Li et al., 1990). Cleavage of the dicistronic precursor by the endonuclease Rnt1 separates snR190 from U14 (Chanfreau et al., 1998). The pre-U3 transcript, by contrast, is produced from a single gene and contains the 7-methyl guanosine (m 7 G) cap structure, which is characteristic of mRNAs, and a short 3Ј extension. During maturation, the m 7 G cap on the pre-U3 is hypermethylated to 2,2,7-trimethylguanosine (TMG) and the 3Ј end is cleaved by Rnt1p, followed by removal of the 3Ј-terminal extension (Kufel et al., 2000). The yeast pre-U3 also contains an intron that is removed by the pre-mRNA splicing machinery, but t...

Nab3’s localization to a nuclear granule in response to nutrient deprivation is determined by its essential prion-like domain

“…The Nab3-Nrd1-containing granule was first identified [26] as a novel structure in or near the nucleus that resembled an RNA quality control site seen in cells with mutated RNA processing factors [59]. Here we observe the reversible induction of Nab3 granules in a PrLD-dependent manner.…”

The Saccharomyces cerevisiae Nrd1-Nab3 Transcription Termination Pathway Acts in Opposition to Ras Signaling and Mediates Response to Nutrient Depletion

“…We believe these starvation-induced, Nrd1-containing nuclear speckles to be novel in S. cerevisiae. Although no similar bodies have been described for wild-type yeast cells, aberrant foci that contain snoRNAs have been described for mutants lacking the functional RNA-processing factors Rna14 and Rna15, and these foci were proposed previously to be RNA control centers in the nucleus (13). We propose that the starvation-induced nuclear speckles formed by Nrd1-GFP could represent similar RNA control centers.…”

Polyadenylation Linked to Transcription Termination Directs the Processing of snoRNA Precursors in Yeast

“…One remaining question is where these processes are localized within the nucleus. In the rrp6Δ mutant, snoRNAs and poly(A) + RNAs accumulate in a discrete nucleolar domain distinct from the nucleolar body (NB) or in nucleoplasmic foci in rrp6Δ/rna14-1 and rrp6Δ/rna15-2 strains ( Carneiro et al., 2007, 2008 ). The authors reason that these spots are not sites of transcription but “surveillance centers” where aberrant RNAs are degraded by the exosome.…”