Cross talk between tRNA and rRNA synthesis in Saccharomyces cerevisiae

Abstract

Temperature-sensitive RNA polymerase III (rpc160-112 and rpc160-270) mutants were analyzed for the synthesis of tRNAs and rRNAs in vivo, using a double-isotopic-labeling technique in which cells are pulse-labeled with [(33)P]orthophosphate and coextracted with [(3)H]uracil-labeled wild-type cells. Individual RNA species were monitored by Northern blot hybridization or amplified by reverse transcription. These mutants impaired the synthesis of RNA polymerase III transcripts with little or no influence on mRNA synthesis but also largely turned off the formation of the 25S, 18S, and 5.8S mature rRNA species derived from the common 35S transcript produced by RNA polymerase I. In the rpc160-270 mutant, this parallel inhibition of tRNA and rRNA synthesis also occurred at the permissive temperature (25 degrees C) and correlated with an accumulation of 20S pre-rRNA. In the rpc160-112 mutant, inhibition of rRNA synthesis and the accumulation of 20S pre-rRNA were found only at 37 degrees C. The steady-state rRNA/tRNA ratio of these mutants reflected their tRNA and rRNA synthesis pattern: the rpc160-112 mutant had the threefold shortage in tRNA expected from its preferential defect in tRNA synthesis at 25 degrees C, whereas rpc160-270 cells completely adjusted their rRNA/tRNA ratio down to a wild-type level, consistent with the tight coupling of tRNA and rRNA synthesis in vivo. Finally, an RNA polymerase I (rpa190-2) mutant grown at the permissive temperature had an enhanced level of pre-tRNA, suggesting the existence of a physiological coupling between rRNA synthesis and pre-tRNA processing.

FIG. 1

Growth properties of conditional mutants defective in RNA polymerase I, II, or III. (A) Conditional (rpa190-2, rpb1-1, rpc160-112, and rpc160-270) mutants and two wild-type (WT) strains (W303-1b and YPH52) were streaked onto YPD plates and incubated for 4 days at 25 and 37°C. Doubling times in liquid medium at 25°C were 140 min (wild-type and rpb1-1 cells), 180 min (rpc160-112 cells), 200 min (rpa190-2 cells), and 250 min (rpc160-270 cells). The pedigrees and genotypes of the corresponding strains are given in Table 1. (B) Growth responses of the rpb1-1, rpa190-2, rpc160-112, and rpc160-270 mutant strains and of the wild-type strain W303-1b in YPD liquid cultures grown exponentially at 25°C and shifted to 37°C for 6 h. Growth was monitored by turbidimetry (see Materials and Methods).

FIG. 2

mRNA synthesis in RNA polymerase I, II, and III mutants shifted to 37°C. Steady-state levels of PEP4, ACT1, NME1, and CYH2 RNAs in RNA polymerase I (rpa190-2), II (rpb1-1), and III (rpc160-112 and rpc160-270) mutants were compared to those in the W303-1b and YPH52 wild-type (WT) strains. ACT1, CYH2, and NME1 RNA levels were determined by Northern hybridization. PEP4 levels were determined by RT-PCR of mutant or wild-type cultures spiked with an aliquot of pep4-Δ wild-type cells (strain OG27GF) (Table 1) expressing a plasmid-borne copy of the GFP gene. The GFP mRNA served as an RNA recovery marker of PEP4 mRNA (see Materials and Methods). Error bars correspond to experimental values obtained in at least two entirely independent RT-PCR or Northern blot experiments, except for NME1 hybridization data (one experiment only). Experimental values were normalized to the wild-type control, arbitrarily taken to have a level of 1.

FIG. 3

tRNA and rRNA synthesis in RNA polymerase I and III mutants. Mutant (rpc160-112, rpc160-270, and rpa190-2) and wild-type (WT) (W303-1b) strains were shifted from 25 to 37°C in low-phosphate medium (YPD*). Cells were labeled in vivo for 10 min with ³³P_i and coextracted with a small amount of tritiated wild-type cells (strain OG27GF grown at 30°C) to provide an interval RNA recovery standard. tRNAs and rRNAs were separated by gel electrophoresis and assayed for ³³P and ³H radioactivity. An example of gel separation is provided (the holes correspond to the recovery of RNA by awl punching, as described in Materials and Methods). Error bars correspond to experimental values obtained in at least two entirely independent in vivo labeling experiments.

FIG. 4

Northern hybridization of RNA polymerase I or III mutants. Northern blot hybridizations with 1 μg of total RNA extracted from wild-type (WT) and mutant cells (same strains as in the previous figures) exponentially grown at 25°C and then shifted to the restrictive temperature (37°C) for 3, 5, and 7 h are shown. The localization data of the pre-rRNA cleavage sites were taken from reference 36). The oligonucleotide probes used specifically hybridized to the 20S pre-rRNA (5′-GCACAGAAATCTCTCACCGT-3′, located between cleavage sites D and A2), 27S pre-rRNA (5′-GCCTAGACGCTCTCTTCTTA-3′, located between cleavage sites C2 and C1 and recognizing all 27S species), 25S rRNA (5′-CCGTGAAATGTTTCTTGGCGTGAG-3′), 18S rRNA (5′-GCCGACGACCGTGGTCTGAAC-3′, internal to the mature 18S sequence), pre-tRNA^Leu3 (5′-CCAAACAACCACTTATTTGTTGA-3′, corresponding to the 5′ leader sequence 19), and mature tRNA^Leu3 (5′-GAACTCTTGCATCTTACGATAC-3′), and SCR1 (5′-CCATCACGGGTCACCT-3′).

FIG. 5

Comparison of pulse-labeling and steady-state levels of tRNA and rRNAs. (A) relative rates of 5S rRNA and tRNA synthesis in RNA polymerase I (rpa190–2) and III (rpc160–112 and rpc160–270) mutants compared to the W303-1b wild type (WT). The data were replotted from Fig. 3. (B) Relative rates of 18S rRNA and tRNA synthesis in RNA polymerase I (rpa190–2) and III (rpc160–112 and rpc160–270) mutants compared to the W303-1b wild type. The data were replotted from Fig. 3. (C) Steady-state levels of 18S rRNA and tRNA synthesis in RNA polymerase I (rpa190–2) and III rpc160–112 and rpc160–270) mutants compared to the wild type (W303-1b). RNA levels were determined by Northern blotting using a 5′-GCCGACGACCGTGGTCTGAAC-3′ internal 18S rRNA probe and a 5′-GAACTCTTGCATCTTACGATAC-3′ internal tRNA^Leu3 probe.