A nucleolar protein related to ribosomal protein L7 is required for an early step in large ribosomal subunit biogenesis

Abstract

The Saccharomyces cerevisiae Rlp7 protein has extensive identity and similarity to the large ribosomal subunit L7 proteins and shares an RNA-binding domain with them. Rlp7p is not a ribosomal protein; however, it is encoded by an essential gene and therefore must perform a function essential for cell growth. In this report, we show that Rlp7p is a nucleolar protein that plays a critical role in processing of precursors to the large ribosomal subunit RNAs. Pulse-chase labeling experiments with Rlp7p-depleted cells reveal that neither 5.8S(S), 5.8S(L), nor 25S is produced, indicating that both the major and minor processing pathways are affected. Analysis of processing intermediates by primer extension indicates that Rlp7p-depleted cells accumulate the 27SA(3) precursor RNA, which is normally the major substrate (85%) used to produce the 5.8S and 25S rRNAs, and the ratio of 27SB(L) to 27SB(S) precursors changes from approximately 1:8 to 8:1 (depleted cells). Because 27SA(3) is the direct precursor to 27SB(S), we conclude that Rlp7p is specifically required for the 5' to 3' exonucleolytic trimming of the 27SA(3) into the 27SB(S) precursor. As it is essential for processing in both the major and minor pathways, we propose that Rlp7p may act as a specificity factor that binds precursor rRNAs and tethers the enzymes that carry out the early 5' to 3' exonucleolytic reactions that generate the mature rRNAs. Rlp7p may also be required for the endonucleolytic cleavage in internal transcribed spacer 2 that separates the 5.8S rRNA from the 25S rRNA.

Figure 1

The pre-rRNA processing pathway in S. cerevisiae. Yeast pre-rRNA is synthesized as a 35S primary transcript that gets cleaved and trimmed at several sites to produce the mature 18S, 5.8S, and 25S rRNAs of the ribosome. Pre-rRNA processing results in a number of recognizable pre-rRNA processing intermediates. The first processing steps occur in the 5′ portion of the 35S RNA and lead to the production of precursors to the small ribosomal subunit RNA; these processing reactions in turn release the 27SA₂ precursor, which will produce the large ribosomal subunit RNAs. The 27SA₂ precursor can enter two different pathways to generate the mature 5.8S and 25S rRNAs. Processing through the major pathway is initiated by RNase MRP cleavage at the A₃ site, and the 27SA₃ RNA gets further trimmed by 5′ to 3′ exonucleases; this exonucleolytic reaction extends to the 5′ end of the 27SB_S precursor (site B_1S), which is also the 5′ end of mature 5.8S_S rRNA. Processing of the 27SA₂ precursor through the minor pathway may also be initiated by 5′ to 3′ exonucleolytic enzyme(s); this reaction extends to site B_1L and generates the 5′ end of the 27SB_L, which is the mature 5′ end of 5.8S_L rRNA. In both the major and minor pathways, endonucleolytic cleavages in ITS2 generate the mature 5′ end of 25S rRNA and the 7S precursors that get trimmed by 3′ to 5′ exonucleases to produce the mature 3′ end of 5.8S rRNAs. This figure has been adapted from Kressler et al. (5).

Figure 2

Rlp7p localizes to the nucleolus. Immunofluorescence microscopy was performed with a strain expressing an HA-tagged Rlp7p. Rabbit anti-HA antibodies were used to detect HA-Rlp7p (A) and the mouse mAb A66 was used to detect the nucleolar protein Nop1p (fibrillarin) (B). Nuclear DNA was stained with 4′,6-diamidin-2-phenylindole (C). The images were merged (D); the yellow staining results from overlapping of the green and red signals.

Figure 3

Rlp7p depletion prevents the synthesis of the 25S rRNA. The pGAL1∷RLP7 strain was depleted of Rlp7p for 17 and 20 h (depleted) and compared with an otherwise isogenic strain, YPH259 (nondepleted). Cells were pulse labeled for 2.5 min with l-[methyl-³H] methionine and then chased for 3 and 12 min with an excess of cold methionine. Total RNA was extracted, and 20,000 cpm per lane was loaded and resolved on a 1.2% formaldehyde–agarose gel. Labeled RNAs were transferred to ζ-probe membrane and visualized by fluorography.

Figure 4

Rlp7p depletion prevents the synthesis of both 5.8S_S and 5.8S_L rRNA. The pGAL1∷RLP7 cells were depleted of Rlp7p for 17 h (depleted) and compared with an otherwise isogenic strain YPH259-pGAD3 (nondepleted). Cells were pulse labeled for 2 min with [5,6-³H] uracil and then chased for 5, 30, and 60 min with an excess of cold uracil. Total RNA was extracted, and 30,000 cpm per lane was resolved on an 8% polyacrylamide gel. Labeled RNAs were transferred to zeta-probe membrane and visualized by fluorography.

Figure 5

The 27SA₃ pre-rRNA accumulates during Rlp7p depletion. RNA was isolated from the pGAL1∷RLP7 strain after growth in galactose (lane 3) or 17 h (lane 4) and 20 h (lane 5) after switching to growth in glucose. RNA was isolated from an otherwise isogenic strain, YPH259, after growth in galactose (lane 2) or 24 h after the switch to growth in glucose (lane 1). Primer extension with the oligonucleotide A₂/A₃ that hybridizes to ITS2 was used to reveal processing sites A₂, A₃, B_1L, and B_1S. Primer extension with the oligonucleotide C₁ that hybridizes to the 25S rRNA was used to map the 5′ end of the 25S rRNA. Products of the primer extension reactions were resolved on polyacrylamide gels next to a DNA sequencing ladder (not shown for 25S) and exposed to x-ray film.

Figure 6

Model for the role of Rlp7p in processing of 27SA precursor RNAs. Rlp7p interacts with the pre-rRNA through its RNA-binding domain and tethers exonucleases (Exo) that carry out the 5′ to 3′ exonucleolytic reactions at the 5′ end of 27SA precursors. Rlp7p could also interact with ITS2 and attract the endonuclease (Endo) that cleaves it, as symbolized by the arrow and question mark.