ERB1, the yeast homolog of mammalian Bop1, is an essential gene required for maturation of the 25S and 5.8S ribosomal RNAs

Abstract

We have recently shown that the mammalian nucleolar protein Bop1 is involved in synthesis of the 28S and 5.8S ribosomal RNAs (rRNAs) and large ribosome subunits in mouse cells. Here we have investigated the functions of the Saccharomyces cerevisiae homolog of Bop1, Erb1p, encoded by the previously uncharacterized open reading frame YMR049C. Gene disruption showed that ERB1 is essential for viability. Depletion of Erb1p resulted in a loss of 25S and 5.8S rRNAs synthesis, while causing only a moderate reduction and not a complete block in 18S rRNA formation. Processing analysis showed that Erb1p is required for synthesis of 7S pre-rRNA and mature 25S rRNA from 27SB pre-rRNA. In Erb1p-depleted cells these products of 27SB processing are largely absent and 27SB pre-rRNA is under-accumulated, apparently due to degradation. In addition, depletion of Erb1p caused delayed processing of the 35S pre-rRNA. These findings demonstrate that Erb1p, like its mammalian counterpart Bop1, is required for formation of rRNA components of the large ribosome particles. The similarities in processing defects caused by functional disruption of Erb1p and Bop1 suggest that late steps in maturation of the large ribosome subunit rRNAs employ mechanisms that are evolutionarily conserved throughout eukaryotes.

Figure 1

Homology of the Erb1/Bop1 primary structure in eukaryotes. Organisms and sequence accession nos are: Sce, S.cerevisiae (NP_013764); Spo, Schizosaccharomyces pombe (S54549); Ath, Arabidopsis thaliana (AAD25679); Cel, Caenorhabditis elegans (T26995); Dme, Drosophila melanogaster (T13579); Mmu, Mus musculus (NP_038509); Hsa, Homo sapiens (BAA09473, incomplete sequence). Initial alignment was obtained with ClustalX (34) and then manually edited using GeneDoc (35). Black shading indicates identical residues and conservative substitutions in all sequences; grey shading indicates such residues in >50% sequences. WD repeats 1–5 were identified by a search against the Pfam database (36) in all seven sequences. The WD1 repeat is the closest to the canonical WD40 structure (5). Numbers within parentheses in each sequence indicate lengths of small unique amino acid insertions and the N-terminal domains, which are not conserved at the primary structure level (see text).

Figure 2

Disruption and rescue of ERB1. (A) Tetrad analysis of an ERB1/erb1::URA3 diploid strain shows 2:2 segregation of viable spores, indicating that ERB1 is essential for viability. (B) Four haploid products from one tetrad obtained by sporulation of an ERB1/erb1::URA3 strain carrying a CEN, HIS3, pGAL1::ERB1 plasmid were streaked on YPGal (top) or YPD (bottom) plates. The Ura and His genotypes of the four clones are indicated at the bottom of the figure, showing that growth of erb1::URA3 haploids is restored by expression of the plasmid-borne, galactose-inducible ERB1.

Figure 3

Effects of Erb1p depletion on growth rate and rRNA. (A) Increase in OD₆₀₀ was measured in parallel in two YDP0135 cultures, one of which was grown in YPGal medium and the other transferred to YPD at time 0. Cultures were periodically diluted to maintain OD₆₀₀ < 1.0 throughout the experiment. (B) Protein lysates were prepared from YDP0135 cells (pGAL1::HA-ERB1) at the indicated times after transfer to YPD and from the control YMS0086 strain (pGAL1::ERB1) (lane c), normalized by protein content, and analyzed by immunoblotting with an anti-HA tag antibody. (C) RNA was extracted from the same cultures as used for protein analysis. Equal amounts of total RNA, as determined by absorbance at 260 nm, were separated on a formaldehyde-containing 1.2% agarose gel, transferred to a nylon membrane and stained with methylene blue.

Figure 4

Pre-rRNA processing in S.cerevisiae. The structure and major sites of processing of the primary 35S pre-rRNA are shown at the top. The external transcribed sequences 5′-ETS and 3′-ETS flank the ends of the mature 18S, 5.8S and 25S rRNA sequences, which are separated by internal transcribed spacers ITS1 and ITS2. Initially, 35S pre-rRNA is cleaved in succession at sites A₀ and A₁ to generate 32S pre-rRNA. The cleavage of 32S pre-rRNA at site A₂ separates the two processing branches that lead to formation of mature rRNAs in the small and large ribosome subunits. The 20S precursor undergoes cleavage at site D after export to the cytoplasm, yielding mature 18S rRNA. The 27SA₂ precursor is processed via two alternative pathways to form the long and short forms of 5.8S rRNA and 25S rRNA. The major pathway, which generates the 5.8S_S form, proceeds through cleavage of 27SA₂ at site A₃ within ITS1 and processing at site B₂, giving rise to 27SA₃, followed by exonucleolytic processing from A₃ to site B_1S, which generates 27SB_S pre-rRNA. In the minor pathway that leads to 5.8S_L formation, 27SB_L pre-rRNA is generated by processing at site B_1L, which occurs by an as yet unidentified mechanism, and processing at B₂. The subsequent processing of both 27SB precursors at sites C₁ and C₂ is probably identical and gives rise to the mature 25S rRNA and 7S pre-rRNAs. The latter are trimmed by exonucleases from the 3′-end to site E, yielding mature 5.8S rRNA species.

Figure 5

Pulse–chase analysis of pre-rRNA processing. YMS0086 cells were grown in galactose-containing synthetic SGal medium (left) or transferred to glucose-containing SD medium for 20 h to deplete Erb1p (center). RNA from the control strain YMS0092 containing wild-type ERB1 is shown on the right. Cells were labeled with l-[methyl-³H]methionine (upper) or [5,6-³H]uracil (lower) for 2 min and chased with non-radioactive methionine or uracil, respectively, for 2, 8 and 16 min as described in Materials and Methods. The same total amount of RNA was loaded on each lane for methyl methionine-labeled samples and the same c.p.m. per lane were loaded for uracil-labeled samples. RNA was separated by formaldehyde–agarose gel electrophoresis, transferred to a nylon membrane and visualized by fluorography.

Figure 6

Northern analysis of pre-rRNA processing effects produced by Erb1p depletion. YDP0135 cells grown in YPGal were transferred to YPD for the time indicated. Equal amounts of total RNA were separated by agarose gel electrophoresis, blotted and hybridized with labeled oligonucleotide probes complementary to different regions of the pre-rRNA transcript (shown at the top). Hybridization of the same membrane with a probe detecting U3 snoRNA, which is not affected by Erb1p depletion, was performed as a loading control. Lane wt, RNA from an isogenic wild-type ERB1 strain YMS0092.

Figure 7

Primer extension analysis of processing in ITS1. Total RNA was isolated from the wild-type ERB1 strain YMS0092 (lane wt) and the pGAL1::HA-ERB1 strain YDP0135 grown on YPGal medium or transferred to YPD medium for the indicated times to deplete Erb1p. Primer extensions were performed on equal amounts of RNA using oligonucleotide y013 (see Fig. 6) as described in Materials and Methods. Positions of primer extension stops (indicated on the right) match the previously determined major processing sites within ITS1 (31); the asterisk indicates an additional stop of unidentified nature observed in this strain.