The final step in the formation of 25S rRNA in Saccharomyces cerevisiae is performed by 5′ → 3′ exonucleases

Abstract

The final stage in the formation of the two large subunit rRNA species in Saccharomyces cerevisiae is the removal of internal transcribed spacer 2 (ITS2) from the 27SB precursors. This removal is initiated by endonucleolytic cleavage approximately midway in ITS2. The resulting 7S pre-rRNA, which is easily detectable, is then converted into 5.8S rRNA by the concerted action of a number of 3'-->5' exonucleases, many of which are part of the exosome. So far the complementary precursor to 25S rRNA resulting from the initial cleavage in ITS2 has not been detected and the manner of its conversion into the mature species is unknown. Using various yeast strains that carry different combinations of wild-type and mutant alleles of the major 5'-->3' exonucleases Rat1p and Xrn1p, we now demonstrate the existence of a short-lived 25.5S pre-rRNA whose 5' end is located closely downstream of the previously mapped 3' end of 7S pre-rRNA. The 25.5S pre-rRNA is converted into mature 25S rRNA by rapid exonucleolytic trimming, predominantly carried out by Rat1p. In the absence of Rat1p, however, the removal of the ITS2 sequences from 25.5S pre-rRNA can also be performed by Xrn1p, albeit somewhat less efficiently.

Yeast Pescadillo is required for multiple activities during 60S ribosomal subunit synthesis

“…The distribution of Rpl25p-eGFP was followed during depletion of Nop7p (Fig+ 3B)+ During growth of the GAL::nop7 strain on RSG medium, Rpl25-eGFP showed the normal, predominantly cytoplasmic distribution+ After transfer to glucose medium for 2 h, increased nuclear staining of Rpl25-eGFP was already visible, and accumulation was strong after 8 h+ The distribution of Rpl25-eGFP fluorescence matched that of DAPI staining, indicating that it was not restricted to the nucleolus+ We conclude that Nop7p is required to allow the export of precursors to the 60S ribosomal subunit from the nucleoplasm to the cytoplasm+ 5+8S S is processed from the 27SB S pre-rRNA, whereas 5+8S L is processed from 27SB L (see Fig+ 1B)+ To determine the levels of the 27SB species, they were analyzed by primer extension (Fig+ 5A) using an oligo hybridizing within the 39 region of ITS2 (oligo 006; see Fig+ 1A)+ Following growth of the GAL::nop7 strain on glucose medium, the level of 27SB S was clearly reduced relative to 27SB L , as shown by the primer extension stops at B 1S and B 1L , respectively (Fig+ 5A, lane 4)+ Consistent with the northern analysis, little alteration was seen in the level of 27SA 2 , as shown by the primer extension stop at site A 2 + In contrast, the level of 27SA 3 , shown by the stop at site A 3 , was substantially elevated+ Using a primer hybridizing within the mature 25S rRNA (oligo 007; see Fig+ 1A), slight accumulation was seen for the primer extension stop at site C 2 , the 59 end of the 26S pre-rRNA (Fig+ 5B)+ This effect was weak, however, and its significance is unclear+ Pulse-chase analysis with [H 3 ]-uracil was performed 16 h after transfer to glucose minimal medium (Fig+ 6)+ Comparison of the wild-type and GAL::nop7 strains showed that accumulation of the 5+8S rRNA was mildly delayed+ Together these data show that depletion of Nop7p resulted in reduced exonuclease digestion from site A 3 to site B 1S + In consequence, the level of the 27SA 3 pre-rRNA was substantially increased, whereas the 27SB S pre-rRNA was depleted together with the 7S S and 6S S pre-rRNAs, leading to reduced accumulation of the mature 5+8S S rRNA+ The 59 end of the 25S rRNA is also generated by exonuclease digestion (Geerlings et al+, 2000;see Fig+ 1B), but this did not appear to be strongly affected, as only a small increase was seen in the primer extension stop at site C 2 + The mild effects on 35S processing are likely to be indirect, as many mutations that inhibit synthesis of 60S subunits result in partial inhibition of the early pre-rRNA processing steps (for further discussion see Venema & Tollervey, 1999)+…”

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

“…The 27SB S and 27SB L pre-rRNAs undergo identical processing by endonucleolytic cleavage at the C 2 site in ITS2 to generate the 25.5S and 7S S or 7S L pre-rRNAs. The 59 end of 25.5S prerRNA is trimmed by Rat1 to form mature 25S rRNA (Geerlings et al 2000). The 39 ends of 7S pre-rRNAs are processed in several steps to produce mature 5.8S S and 5.8S L rRNAs differing by 6 nt at their 59 ends Mitchell et al 1996).…”

“…The 59-39 exonucleases Rat1 and Rrp17 rapidly process 25.5S pre-rRNA to 25S rRNA (Geerlings et al 2000). Trimming of 7S pre-rRNA to 5.8S S or 5.8S L rRNAs requires at least four steps and a number of different nucleases and occurs sequentially in the nucleus and then the cytoplasm (Henry et al1994;Tollervey 2005, 2010).…”

mRNA stability in the nucleus

“…It is required after cleavage and polyadenylation of nascent transcripts to degrade the downstream cleaved RNA from 5'-3' direction (Richard and Manley, 2009). Rat1p is also involved in the process of rRNA maturation: ITS1, one of the internal transcribed spacers (ITS1 and ITS2), locates at the flanking site of 5.8S rRNA, which is trimmed by Rat1p from the 5' end of the rRNA precursor after endoribonuclease cleavage (Henry et al, 1994;Geerlings et al, 2000). Rat1p recently has been suggested to play an important role in telomere maintenance by degrading telomeric repeat-containing RNA (TERRA), which is a long non-coding RNA that represses telomerase activity, and thus activates telomere elongation (Luke et al, 2008).…”