Yeast 18S rRNA dimethylase Dim1p: a quality control mechanism in ribosome synthesis?

Abstract

One of the few rRNA modifications conserved between bacteria and eukaryotes is the base dimethylation present at the 3' end of the small subunit rRNA. In the yeast Saccharomyces cerevisiae, this modification is carried out by Dim1p. We previously reported that genetic depletion of Dim1p not only blocked this modification but also strongly inhibited the pre-rRNA processing steps that lead to the synthesis of 18S rRNA. This prevented the formation of mature but unmodified 18S rRNA. The processing steps inhibited were nucleolar, and consistent with this, Dim1p was shown to localize mostly to this cellular compartment. dim1-2 was isolated from a library of conditionally lethal alleles of DIM1. In dim1-2 strains, pre-rRNA processing was not affected at the permissive temperature for growth, but dimethylation was blocked, leading to strong accumulation of nondimethylated 18S rRNA. This demonstrates that the enzymatic function of Dim1p in dimethylation can be separated from its involvement in pre-rRNA processing. The growth rate of dim1-2 strains was not affected, showing the dimethylation to be dispensable in vivo. Extracts of dim1-2 strains, however, were incompetent for translation in vitro. This suggests that dimethylation is required under the suboptimal in vitro conditions but only fine-tunes ribosomal function in vivo. Unexpectedly, when transcription of pre-rRNA was driven by a polymerase II PGK promoter, its processing became insensitive to temperature-sensitive mutations in DIM1 or to depletion of Dim1p. This observation, which demonstrates that Dim1p is not directly required for pre-rRNA processing reactions, is consistent with the inhibition of pre-rRNA processing by an active repression system in the absence of Dim1p.

FIG. 1

Structure of yeast 35S pre-rRNA and the pre-rRNA processing pathway. (A) 35S pre-rRNA. The sequences encoding the mature 18S, 5.8S, and 25S rRNAs are embedded in the 5′ and 3′ external transcribed spacers (ETS) and in ITS1 and ITS2. Sites of pre-rRNA processing are indicated with uppercase letters (A₀ to E), and the oligonucleotides used for Northern hybridization and primer extension are indicated with lowercase letters (a to h). The site of dimethylation is denoted as m₂⁶A. (B) Pre-rRNA processing pathway. Processing of the 35S primary transcript starts at site A₀ in the 5′ ETS. The resulting 33S pre-rRNA is processed at sites A₁ and A₂, giving rise successively to the 32S pre-rRNA and to the 20S and 27SA₂ precursors. Cleavage at site A₂ separates the pre-rRNAs destined for the small and large ribosomal subunits. The 20S precursor is dimethylated and endonucleolytically cleaved at site D to yield mature 18S rRNA. Cleavage of 27SA₂ at site A₃, by RNase MRP, is rapidly followed by exonucleolytic digestion to site B1_S, generating the 27SB_S precursor. Mature 25S rRNA and 7S pre-rRNA are released from 27SB_S following cleavages at sites C₁ and C₂. 7S pre-rRNA undergoes a rapid 3′→5′ exonuclease digestion to site E, generating the mature 3′ end of 5.8S rRNA (not represented). For simplicity, only the major processing pathway, from 27SA₂ to 5.8S_S and 25S rRNA, is shown; an alternative pathway generates the minor 5.8S_L rRNA, which is 7 to 9 nucleotides 5′ extended. The steps that require Dim1p are indicated. (C) Pre-rRNA processing in dim1 TS strains. 35S pre-rRNA is cleaved normally at site A₀. 33S pre-rRNA accumulates and is cleaved at site A₃, providing the 27SA₃ that is normally processed to 5.8S_S and 25S and the aberrant 22S pre-rRNA that is not dimethylated and not processed to 18S rRNA.

FIG. 2

Conditional growth phenotypes of dim1 TS strains. Dilutions (1× to 10²×) of strains dim1-1 to dim1-10, along with the wild-type isogenic DIM1 control (strain YDL209) (W.T.), were spotted on minimal plates at 18, 25, 30, and 37°C and incubated for 3 days. Most of the strains are very sensitive to elevated temperatures; some are also slightly CS (dim1-1 and dim1-7).

FIG. 3

Mapping of dim1 TS mutations. Schematic representation of Dim1p (318 residues) drawn to scale. The S-adenosylmethionine binding domain (SAM), the putative catalytic residues (NxPY), and the 32 point mutations (asterisks) identified in the 10 dim1 TS alleles are represented.

FIG. 4

Pre-rRNA processing in dim1 TS strains. (A) Probe against the 5′ region of ITS1 (oligonucleotide e [Fig. 1A]). (B) Probes against mature 18S and 25S rRNA (oligonucleotides a and f [Fig. 1A]). RNA was extracted from the DIM1 (strain YDL209) (W.T.) and dim1 TS strains following growth at 23°C (0-h lanes) and at intervals following transfer to 37°C (2-, 8-, and 23-h lanes) and separated on 1.2% agarose-formaldehyde gels. The 22S pre-rRNA extends from site A₀ to site A₃ and results from the inhibition of cleavages at sites A₁ and A₂ in the dim1 TS strains. At the late time point of transfer to 37°C (23 h), the normally minor and barely detectable 23S pre-rRNA that extends from the 5′ end of the primary transcript to site A₃ accumulates to the same levels in the wild-type and dim1 TS strains. In this experiment, all samples, including the wild-type control, showed some accumulation of 35S pre-rRNA and 23S RNA after 23 h at 37°C; this was not observed in other experiments.

FIG. 5

Overall level of dimethylation in dim1 TS strains. RNA was extracted from the DIM1 (strain YDL209) (W.T.) and dim1 TS strains following growth at 23°C (0-h lanes) and at intervals following transfer to 37°C (2-, 8-, and 23-h lanes) and analyzed by primer extension with oligonucleotide e (Fig. 1A). The positions of primer extension stops due to the presence of the modifications are indicated. A DNA sequence made with the same primer is shown as a size marker.

FIG. 6

Levels of nondimethylated 18S rRNA in dim1 TS strains. (A) RNA was extracted from the DIM1 (strain YDL209) (W.T.) and dim1 TS strains following growth at 23°C (0-h lanes) and at intervals following transfer to 37°C (2-, 8-, and 23-h lanes) and analyzed by primer extension with oligonucleotide d, which is complementary to the very 3′ end of 18S rRNA (Fig. 1A). The reactions were performed with dideoxyadenosine nucleotides in place of deoxyadenosine. The site of priming is 3 nucleotides 3′ to A₁₇₈₀, and no A residues are incorporated before the site of modification (see panel B). Dimethylation of A₁₇₇₉ A₁₇₈₀ blocks primer extension. Extensions carried out on nondimethylated rRNA extend through the A₁₇₇₉ A₁₇₈₀ site but are blocked 2 nucleotides 5′ to A₁₇₇₉ (position U₁₇₇₇). The positions of primer extension stops due to the presence of the modifications are indicated (a, b and c). A DNA sequence made with the same primer is shown as a size marker. Lanes 25 to 27, control lanes (lane 25, no RNA [P denotes primer alone]; lane 26, same as lane 4; lane 27, RNA extracted from the GAL::dim1 strain (strain YDL302) following transfer to glucose for 60 h). (B) Schematic representation of the 3′ end of 18S rRNA. Upper line, rRNA strand (the thick line represents the last 16 nucleotides). Lower line, complementary cDNA strand (the thick broken line represents oligonucleotide d). The three potential extended products are represented by thin lines (a, b, and c). The positions of the primer extension stops due to the presence of the modifications are indicated as a and b; the position of the primer extension stop due to read-through of the dimethylation site is indicated as c.

FIG. 7

18S and 25S accumulation in dim1 TS strains expressing rDNA from different promoters. Lane 1, RNA extracted from a DIM1 wild-type strain; lane 2, RNA extracted from a DIM1 wild-type strain also expressing pre-rRNA from a GAL promoter; lanes 3 and 4, RNA extracted from a dim1-1 strain also expressing pre-rRNA from a GAL promoter; lanes 5 and 6, RNA extracted from a dim1-1 strain also expressing pre-rRNA from a PGK promoter. The same Northern filter was hybridized with probes complementary to the 25S and 18S rRNAs. The probes used are specific either for the tagged rRNAs synthesized from the RNA Pol II promoter or for the nontagged rRNAs transcribed from the chromosomal rDNA (RNA Pol I).

FIG. 8

pPGK::rDNA transcripts are insensitive to Dim1p depletion. (A) Probes specific to mature 25S and 18S rRNA produced from the pPGK::rDNA construct (oligonucleotides b and g [Fig. 1A]). (B) Probes specific to the mature 25S and 18S rRNA produced from the chromosomal rDNA units (oligonucleotides c and h [Fig. 1A]). RNA was extracted from the GAL::dim1 strain transformed with the pPGK::rDNA construct following growth in galactose (0-h lanes) and at intervals following transfer to glucose (2- to 60-h lanes) and separated on a 1.2% agarose-formaldehyde gel.

FIG. 9

In vitro translation analysis of dim1 TS strains. Cytoplasmic S30 extracts of dim1-2 strains (YDL321-1 and YDL321-2), a dim1-1 strain (YDL324), and the wild-type (W.T.) isogenic control (strain MBS) were prepared following growth at 23°C. Standardized amount of extracts were incubated with 0, 2, and 10 ng of preprolactin mRNA (A) or 0, 20, and 100 ng of CAT mRNA (B and D). YDL321-1 and YDL321-2 are two independently isolated integrants of the dim1-2 allele in the MBS strain (see Materials and Methods and Table 1). The dim1-2 strain used in panel D is YDL321-2. Translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are indicated at their expected lengths (11 and 25 kDa). (C) 18S rRNA dimethylation in cell extracts, analyzed as described in the legend to Fig. 6. RNA was extracted from strains YDL321-1, YDL321-2, and the wild-type strain (strain MBS) following growth at 23°C. Lanes 1 to 3, control lanes (lane 1, no RNA [P denotes primer alone]; lane 2, RNA extracted from strain MBS following transfer to 37°C for 23 h; lane 3, RNA extracted from the GAL::dim1 strain [strain YDL302] following transfer to glucose for 60 h).

FIG. 10

Antibiotic sensitivities of dim1 TS strains. Dilutions (1× and 10×) of dim1-1 and dim1-2 strains, along with the isogenic wild-type (W.T.) DIM1 control strain, were spotted on complete medium supplemented with paromomycin and neomycin B at the concentrations indicated. Plates were incubated at 23°C.

FIG. 11

Subcellular localization of Dim1p. Indirect immunofluorescence with strain YDL102A (Dim1p-3× Myc). Cells were incubated with either anti-Nop1p (α-Nop1p) or anti-Myc (α-Myc) antibody followed by Texas red (TR) and DAPI staining. Both primary antibodies are mouse monoclonal antibodies; therefore, cells were labelled independently.