All three functional domains of the large ribosomal subunit protein L25 are required for both early and late pre-rRNA processing steps in Saccharomyces cerevisiae

Abstract

Mutational analysis has shown that the integrity of the region in domain III of 25S rRNA that is involved in binding of ribosomal protein L25 is essential for the production of mature 25S rRNA in the yeast Saccharomyces cerevisiae. However, even structural alterations that do not noticeably affect recognition by L25, as measured by an in vitro assay, strongly reduced 25S rRNA formation by inhibiting the removal of ITS2 from the 27S(B) precursor. In order to analyze the role of L25 in yeast pre-rRNA processing further we studied the effect of genetic depletion of the protein or mutation of each of its three previously identified functional domains, involved in nuclear import (N-terminal), RNA binding (central) and 60S subunit assembly (C-terminal), respectively. Depletion of L25 or mutating its (pre-)rRNA-binding domain blocked conversion of the 27S(B) precursor to 5.8S/25S rRNA, confirming that assembly of L25 is essential for ITS2 processing. However, mutations in either the N- or the C-terminal domain of L25, which only marginally affect its ability to bind to (pre-)rRNA, also resulted in defective ITS2 processing. Furthermore, in all cases there was a notable reduction in the efficiency of processing at the early cleavage sites A0, A1 and A2. We conclude that the assembly of L25 is necessary but not sufficient for removal of ITS2, as well as for fully efficient cleavage at the early sites. Additional elements located in the N- as well as C-terminal domains of L25 are required for both aspects of pre-rRNA processing.

Figure 1

The S.cerevisiae rDNA unit and formation of the mature rRNA species. (A) Genetic organization of the rDNA unit. Black bars correspond to the mature sequences, lines to the external and internal spacer sequences. The processing sites are indicated, as are the positions of the various probes used for the northern and primer extension analyses. (B) The rRNA processing pathway in S.cerevisiae. Vertical arrows correspond to endonucleolytic cleavages, kinked arrows to exonucleolytic processing. (C) Sequences of the probes depicted in (A).

Figure 2

Effect of depletion or mutation of L25 on accumulation of the mature rRNA species. YCR61 cells were transformed with the YCplac111 plasmid carrying the wild-type L25 gene (lanes 1 and 2), an ‘empty’ vector (lanes 3 and 4) or a vector containing one of several mutant forms of the L25 gene (lanes 5–14). Tranformants were grown on galactose-based medium and shifted to glucose-based medium. Total RNA was isolated immediately before and 24 h after the shift and analyzed by northern hybridization using oligonucleotides complementary to 25S rRNA (probe G, A), 18S rRNA (probe F, B) and 5.8S rRNA (probe H, C). See Figure 1 for location of the probes. SΔN, replacement of the 41 N-terminal amino acids with the NLS of the SV40 large T antigen (34); M3 and M5, mutations in the conserved motif at the C-terminus of the RNA-binding domain (M3, L₁₂₆→K; M5, K₁₂₀K₁₂₁→RR; 33,36); ΔC4, deletion mutant lacking the four C-terminal amino acids; L₁₃₅→R, point mutation in the C-terminal functional element of L25 (36).

Figure 3

Effect of depletion or mutation of L25 on pre-rRNA processing. YCR61 cells were transformed with the YCplac111 plasmid carrying the wild-type L25 gene (lanes 1 and 2), an ‘empty’ plasmid (lanes 3 and 4) or a vector containing one of several mutant forms of the gene (lanes 5–14; see legend to Fig. 2 for details). Transformants were grown on galactose-based medium and shifted to glucose-based medium. Total RNA was isolated immediately before and 24 h after the shift and analyzed by northern hybridization with oligonucleotides complementary to different spacer regions to determine the levels of the various pre-rRNA intermediates using the same blot as shown in Figure 2. (A) Probe A; (B) probe D; (C) probe C; (D) probe C; (E) probe E. The pre-rRNA species detected is indicated in each of the different panels. See Figure 1 for location and sequence of the probes.

Figure 4

Effect of depletion or mutation of L25 on the accumulation of 7S pre-rRNA. YCR61 cells transformed with YCplac111 carrying the wild-type L25 gene (lanes 1–3), an ‘empty’ plasmid (lanes 4–6) or a plasmid carrying the M3 (lanes 7–9) or L₁₃₅→R mutant forms of L25 (lanes 10–12), respectively, were shifted from galactose- to glucose-based medium (see legend to Fig. 2 for details). Total RNA was isolated immediately before and at different times after the shift, separated on an 8% polyacrylamide gel and subjected to northern analysis using probe D complementary to the upstream region of ITS2 (see Fig. 1).

Figure 5

Effect of depletion or mutation of L25 on accumulation of the A₀–A₁ spacer fragment. YCR61 cells transformed with YCplac111 containing the wild-type L25 gene (lanes 1 and 2), the ‘empty’ plasmid (lanes 3 and 4) or a plasmid carrying the M3 (lanes 5 and 6) or L₁₃₅→R (lanes 7 and 8) mutant forms of the L25 gene, respectively, were shifted from galactose- to glucose-based medium (see legend to Fig. 2 for details). Total RNA was isolated immediately before and 24 h after he shift, separated on an 8% polyacrylamide gel and subjected to northern analysis using probe B complementary to the 5′-ETS downstream from site A₀ (see Fig. 1).

Figure 6

Effect of depletion of L25 on pre-rRNA processing. YCR61 cells transformed with the empty YCplac111 plasmid were shifted from galactose- to glucose-based medium. Total RNA was isolated immediately before (lane 1) and 3 h after (lane 2) the shift. The RNA was subjected to northern analysis using probe E complementary to the region of ITS1 upstream from site A₂.

Figure 7

Effect of depletion of L25 on cleavage at different processing sites. YCR61 cells transformed with the YCplac111 plasmid either with the wild-type L25 gene (lanes 1 and 2) or without an insert (lanes 3 and 4) were shifted from galactose- to glucose-based medium. Total RNA was isolated immediately before (lanes 1 and 3) and 24 h after (lanes 2 and 4) the shift. The RNA was subjected to primer extension analysis. (A and B) Probe F; (C–E) probe H. See Figure 1 for location of the probes. The site detected in each of the different panels is indicated.