The RNA catabolic enzymes Rex4p, Rnt1p, and Dbr1p show genetic interaction with trans-acting factors involved in processing of ITS1 in Saccharomyces cerevisiae pre-rRNA

Abstract

Eukaryotes have two types of ribosomes containing either 5.8SL or 5.8SS rRNA that are produced by alternative pre-rRNA processing. The exact processing pathway for the minor 5.8SL rRNA species is poorly documented. We have previously shown that the trans-acting factor Rrp5p and the RNA exonuclease Rex4p genetically interact to influence the ratio between the two forms of 5.8S rRNA in the yeast Saccharomyces cerevisiae. Here we report a further analysis of ITS1 processing in various yeast mutants that reveals genetic interactions between, on the one hand, Rrp5p and RNase MRP, the endonuclease required for 5.8SS rRNA synthesis, and, on the other, Rex4p, the RNase III homolog Rnt1p, and the debranching enzyme Dbr1p. Yeast cells carrying a temperature-sensitive mutation in RNase MRP (rrp2-1) exhibit a pre-rRNA processing phenotype very similar to that of the previously studied rrp5-33 mutant: ITS2 processing precedes ITS1 processing, 5.8SL rRNA becomes the major species, and ITS1 is processed at the recently reported novel site A4 located midway between sites A2 and A3. As in the rrp5-Delta3 mutant, all of these phenotypical processing features disappear upon inactivation of the REX4 gene. Moreover, inactivation of the DBR1 gene in rrp2-1, or the RNT1 gene in rrp5-Delta3 mutant cells also negates the effects of the original mutation on pre-rRNA processing. These data link a total of three RNA catabolic enzymes, Rex4p, Rnt1p, and Dbr1p, to ITS1 processing and the relative production of 5.8SS and 5.8SL rRNA. A possible model for the indirect involvement of the three enzymes in yeast pre-rRNA processing is discussed.

FIGURE 1.

Pre-rRNA processing in Saccharomyces cerevisiae. (A) Structure of the rDNA transcription unit, including the various processing sites. (B) The pre-rRNA processing pathway. The processing steps in ITS1 involving RNase MRP and the 5′ → 3′ exonucleases Xrn1p and Rat1p are indicated. (C) Enlargement of the central portion of the pre-rRNA showing the positions of the different probes used for Northern and primer extension analysis. See Materials and Methods for the sequences of the probes.

FIGURE 2.

5.8S rRNA levels in the D308 (rrp2-1; lanes 2,3) and FVY010A (rrp2-1/rex4-null; lanes 4,5) mutant strains. Cultures of the two strains were grown in liquid YPD medium at 23°C to midexponential phase and then shifted to 37°C. Total RNA was isolated from cells harvested immediately before (lanes 2,4) and 8 h after (lanes 3,5) the shift and analyzed on an 8% polyacrylamide gel. The gel was stained with ethidium bromide and the relative levels of 5.8S_S and 5.8S_L rRNA were determined by scanning.

FIGURE 3.

Northern analysis of high-molecular-weight pre-rRNA isolated from wild-type YJV159 cells (lanes 1,2), strains D308 (rrp2-1; lanes 3,4), and FVY010A (rrp2-1/rex4-null; lanes 5,6). The strains were grown on YPD medium and shifted from 23°C to 37°C at midexponential phase. Total RNA was isolated from cells harvested immediately before (L) and 8 h after (H) the shift, separated on a 1.2% agarose gel, and subjected to Northern analysis using probes 5 (A) and 3 (B).

FIGURE 4.

Northern analysis of low-molecular-weight processing intermediates in strains D308 (rrp2-1; lanes 2,3) and FVY010A (rrp2-1/rex4-null; lanes 4,5). Cells were grown on YPD medium and shifted from 23°C to 37°C at midexponential phase. Total RNA was isolated from cells harvested immediately before (L) and 8 h after (H) the shift, separated on an 8% poly-acrylamide gel, and subjected to Northern analysis. (A–E) Northern hybridization with the various probes as indicated. The same gel was hybridized sequentially with probes 4, 3, 1, 2, and 5. Lane 1 contains RNA from exponential growing wild-type cells (YJV159) grown at 23°C. The lower portion of D shows a shorter exposure of the region containing the 5.8S rRNA bands of same blot. (F) Northern hybridization using probe 3 of total RNA isolated from rrp2-1 mutant cells at either 23°C (L) or 37°C (H) and separated on a 1.2% agarose gel to visualize the relative levels of the 27SA₂ pre-rRNA and the A2-E fragment.

FIGURE 5.

Primer extension analysis of pre-rRNA isolated from strains D308 (rrp2-1; lanes 3,4) and FVY010A (rrp2-1/rex4-null; lanes 5,6). Total RNA was isolated from cells immediately before (L) and 8 h after (H) the shift to the nonpermissive temperature and analyzed by primer extension using probe 4 (A) or probe 5 (B). The positions of the stops corresponding to the different processing sites are indicated. Lanes 1 and 2 show the controls using RNA from wild-type YJV159 cells treated in the same manner. The band indicated by the asterisk represents an artificial stop.

FIGURE 6.

Effect of inactivation of the RNT1 gene on growth and pre-rRNA processing in cells dependent upon the Rrp5-Δ3p mutant protein. Strain YJV204 [Gal::rrp5/RNT1/p(rrp5-Δ3)] and FVY07A [Gal::rrp5/rnt1::KAN/p(rrp5-Δ3)] were grown on galactose-based medium to midexponential phase and plated in 10-fold serial dilutions on either galactose or glucose plates (A). The same strains were grown on galactose-based medium and shifted to glucose-based medium. Samples were taken immediately before and 8 h after the shift, either directly (- LiCl) or after treatment of the culture with 0.2 M LiCl for 1 h (+ LiCl). Total RNA was isolated and subjected to Northern analysis using probe 4 (B) or probe 2 (C) or primer extension analysis using probe 4 (D).

FIGURE 7.

In vitro analysis of the cleavage of model substrates representing the Rnt1p cleavage site in the 3′-ETS or site A4, respectively. (A) Structural comparison of the model substrates. Conserved nucleotides are boxed. The shaded box highlights the nucleotides in the ITS1 stem that are strongly conserved among various yeast strains (Eppens et al. 2002). (B) Transcripts corresponding to the model substrates were prepared by in vitro transcription, 5′-end labeled, and tested for cleavage by incubating them with 0.8 pmol of recombinant Rnt1p in the presence of two different concentrations of KCl. Cleavage products were fractionated on 20% denaturing polyacrylamide gels. The RNA molecular weight marker is shown on the left. The positions of the substrate (S) and product (P) are indicated.

FIGURE 8.

Effect of inactivation of the DBR1 gene on pre-rRNA processing in rrp2-1 mutant cells. Cultures of YJV159 (wild-type; lanes 1,2), Y04999 (dbr1-null; lanes 3,4), D308 (rrp2-1; lanes 5,6), and FVY20 (rrp2-1/dbr1-null; lanes 7,8) were grown in liquid YPD medium at 23°C to midexponential phase and then shifted to 37°C. Total RNA was isolated from cells harvested immediately before (L) and 8 h after (H) the shift and analyzed on an 8% polyacrylamide gel. (A) Northern analysis using a probe complementary to U24 snoRNA. (B) EtBr staining to visualize 5.8S rRNA. (C) Northern analysis using probe 2. The position of the 5.8S rRNA species as determined by EtBr staining is indicated

FIGURE 9.

Model explaining the effect of the various mutations on ITS1 processing in yeast. For reasons of clarity only the postulated snoRNP and not the remainder of the processing/assembly machinery is shown. The position of the snoRNP is arbitrary and does not necessarily indicate its direct interaction with, or position relative to, the spacer. The type size of the processing steps indicated at the right indicates their approximate relative frequency. See text for further details.