Functional significance of intermediate cleavages in the 3'ETS of the pre-rRNA from Schizosaccharomyces pombe

Abstract

Pathways for the maturation of ribosomal RNAs are complex with numerous intermediate cleavage sites that are not always conserved closely in the course of evolution. Both in eukaryotes and bacteria genetic analyses and in vitro studies have strongly implicated RNase III-like enzymes in the processing of rRNA precursors. In Schizosacharomyces pombe, for example, the RNase III-like Pac1 nuclease has been shown to cleave the free 3'ETS at two known intermediate sites but, in the presence of RAC protein, the same RNA also is cleaved at the 3'-end of the 25 S rRNA sequence. In this study normal and mutant 3'ETS sequences were digested with the Pac1 enzyme to further evaluate its role in rRNA processing. Accurate cleavage at the known intermediate processing sites was dependent on the integrity of the helical structure at these sites as well as a more distal upper stem region in the conserved extended hairpin structure of the 3'ETS. The cleavage of mutant 3'ETS sequences also generally correlated with the known effects of these mutations on rRNA production, in vivo. One mutant, however, was efficiently processed in vivo but was not a substrate for the Pac1 nuclease, in vitro. In contrast, in the presence of RAC protein, the same RNA remained susceptible to Pac1 nuclease cleavage at the 3'-end of the 25 rRNA sequence, indicating that the removal of the 3'ETS does not require cleavage at the intermediate sites. These results suggest that basic maturation pathways may be less complex than previously reported raising similar questions about other intermediate processing sites, which have been identified by analyses of termini, and/or processing, in vitro.

Figure 1

Pac1 nuclease cleavage of a 5′-labeled 3′ETS substrate from the S.pombe pre-rRNA. A normal 3′ETS substrate was prepared in vitro by transcription with T₇ RNA polymerase, labeled at the 5′-end (Ctl) and aliquots (10 000 c.p.m.) were digested with 0.4 (a), 1.2 (b) or 3.6 ng (c and d) of Pac1 RNase in the absence (a–c) and presence (d) of 100 ng of unlabeled 3′ETS substrate. Digests were fractionated on a 10% denaturing polyacrylamide gel and the fragments were detected by autoradiography. Labeled substrate, which was partially digested with T1, ribonuclease (T₁) or mild base (B) was included as nucleotide markers. The positions of known intermediate processing sites (13) are indicated on the right.

Figure 2

Pac1 nuclease cleavage of mutant 5′-labeled 3′ETS substrates from S.pombe pre-rRNA. Normal or mutant 3′ETS substrates were prepared in vitro by transcription with T₇ RNA polymerase, labeled at the 5′-end and aliquots (10 000 c.p.m.) were digested with 3.6 ng of Pac1 RNase (8.0 nM) in the presence 100 ng of unlabeled normal 3′ETS substrate. Undigested normal 3′ETS substrate (Ctl) and digests of normal 3′ETS (Wt), 3′ETSG₃₉G₄₁G₄₂ (a), 3′ETSΔ22 (b), 3′ETSΔ12 (c), 3′ETSG₃₉G₄₁G₄₂ G₆₈C₇₂C₈₁C₈₂C₈₄ (e), 3′ETSG₃₉G₄₁G₄₂C₈₁C₈₂C₈₄ (f), 3′ETSC₇₀A₇₂U₇₃C₈₁ C₈₂C₈₄ (g), 3′ETSC₇₀A₇₂U₇₃ (h), 3′ETSA₂₇A₂₈C₃₉ (i) and 3′ETSG₈₆ G₈₇G₈₈G₈₉G₉₀ (j) were fractionated on a 10% denaturing polyacrylamide gel and the fragments were detected by autoradiography. Labeled substrate which was partially digested with T1 ribonuclease (T1) or mild base (B) was included as nucleotide markers. The position of the known intermediate processing sites at U₊₄₁ (13) is indicated on the right. The mutant lanes are labeled to correspond with the structural estimates presented in Figures 3 and 4.

Figure 3

Effect of the upper hairpin region on Pac1 nuclease cleavage of the 3′ETS in S.pombe pre-rRNA. Normal or mutant 3′ETS substrates were prepared in vitro, labeled at the 5′-end and digested with Pac1 RNase in the presence of 100 ng of unlabeled normal RNA; the digests were fractionated as described for Figures 1 and 2. The degree of digestion, indicated as an average for three experiments, was determined as a percentage of the digestion with the normal 3′ETS substrate. The changed residues are indicated by shading and deleted regions are indicated by the number of residues. The normal structure is shown on the left as previously reported (14). The amounts of plasmid-derived 25S rRNA, as determined by S1 mapping previously (19) or in the course of this study, are indicated as strong band (++) or none detectable (n.d.), respectively; the amount of plasmid-derived 5.8S rRNA as a percentage of that observed with a normal 3′ETS structure was taken from the same study or determined by electrophoretic fractionation.

Figure 4

Effect of the lower hairpin region on Pac1 nuclease cleavage of the 3′ETS in S.pombe pre-rRNA. Normal or mutant 3′ETS substrates were prepared in vitro, labeled at the 5′-end and digested with Pac1 RNase in the presence of 100 ng of unlabeled normal RNA; the digests were fractionated as described for Figures 1 and 2. The degree of digestion, indicated as an average for three experiments, was determined as a percentage of the digestion with the normal 3′ETS substrate. The changed residues are indicated by shading and deleted regions are indicated by the number of residues. The normal structure is shown on the left as previously reported (14). The amounts of plasmid-derived 25S rRNA, as determined by S1 mapping previously (19) or in the course of this study, are indicated as strong band (++) or weak band (+), respectively; the amount of plasmid-derived 5.8S rRNA as a percentage of that observed with a normal 3′ETS structure was taken from the same study or determined by electrophoretic fractionation.

Figure 5

RAC protein-induced Pac1 nuclease cleavage at the 3′-end of the 25S rRNA sequence in S.pombe pre-rRNA. Normal (left panel) or mutant (3′ETSG₈₆G₈₇G₈₈G₈₉G₉₀) RNA substrate (middle panel) consisting of the last 55 nucleotides in the mature 25S rRNA and the conserved extended hairpin structure in the S.pombe 3′ETS was prepared in vitro by transcription with T₇ RNA polymerase, labeled at the 5′-end (3′ETS) and aliquots (10 000 c.p.m.) were digested with Pac1 RNase in the absence (Pac) and presence (Pac + RAC) of ∼250 ng of RAC protein extract. Digests were fractionated on a 10% denaturing polyacrylamide gel and the fragments were detected by autoradiography. Labeled substrate that was partially digested with T1 ribonuclease (T1) or mild base (OH^–) was included as nucleotide markers. The position of the known PAC1 intermediate cleavage site as described in Figure 1 and the mature 3′-end of the 25S rRNA are indicated on the right. Termini in the 3′ETS region of RNA from S.pombe cells expressing the same mutant RNA also were mapped with S1 nuclease (right panel) as described previously described (14,19) and fractionated on a 6% denaturing polyacrylamide gel. The positions of the known termini, based on standard dideoxy sequencing reactions as residue markers, are indicated on the right.