Functional characterization of the conserved amino acids in Pop1p, the largest common protein subunit of yeast RNases P and MRP

Abstract

RNase P and RNase MRP are ribonucleoprotein enzymes required for 5'-end maturation of precursor tRNAs (pre-tRNAs) and processing of precursor ribosomal RNAs, respectively. In yeast, RNase P and MRP holoenzymes have eight protein subunits in common, with Pop1p being the largest at >100 kDa. Little is known about the functions of Pop1p, beyond the fact that it binds specifically to the RNase P RNA subunit, RPR1 RNA. In this study, we refined the previous Pop1 phylogenetic sequence alignment and found four conserved regions. Highly conserved amino acids in yeast Pop1p were mutagenized by randomization and conditionally defective mutations were obtained. Effects of the Pop1p mutations on pre-tRNA processing, pre-rRNA processing, and stability of the RNA subunits of RNase P and MRP were examined. In most cases, functional defects in RNase P and RNase MRP in vivo were consistent with assembly defects of the holoenzymes, although moderate kinetic defects in RNase P were also observed. Most mutations affected both pre-tRNA and pre-rRNA processing, but a few mutations preferentially interfered with only RNase P or only RNase MRP. In addition, one temperature-sensitive mutation had no effect on either tRNA or rRNA processing, consistent with an additional role for RNase P, RNase MRP, or Pop1p in some other form. This study shows that the Pop1p subunit plays multiple roles in the assembly and function of of RNases P and MRP, and that the functions can be differentiated through the mutations in conserved residues.

FIGURE 1.

Alignment of the four conserved regions (COR1–4) of Pop1p from S. cerevisiae (scPop1p) and its homologs from S. pombe (spPop1), Drosophila (dPop1), Anopheles (aPop1), mouse (mPop1), and human (hPop1). The sequences were aligned with the Clustal W algorithm. Identical amino acids are highlighted in black with white letters, whereas similar amino acids that occupy homologous positions are highlighted in gray. Less conserved positions that were also mutated are underlined. Positions of the amino acids in each Pop1 protein are indicated with numbers flanking the corresponding sequence. The alignment has confirmed the previously identified COR1, -2, and -4 (Lygerou et al. 1996b), and uncovered another conserved region, COR3.

FIGURE 2.

Growth of the 3HA-Pop1p mutants on synthetic minimum medium. The strain background for this test was SXY1. (A) Growth at 30°C and 37°C on solid medium. Position of each strain is illustrated in the right panel. Strain SXY1/M₂₅₆, indicated with an asterisk, is ts for growth on plates, but the growth in liquid medium at 37°C is only mildly slow compared to the wild-type control strain (shown in B). (B) Growth curves of representative strains at 37°C in liquid medium. The growth curves of strains 3HA-Pop1 (control) and M₂₅₆ are indicated on the plot.

FIGURE 3.

Northern blot analysis of pre-tRNA^Leu processing in representative 3HA-pop1 mutants at 30°C and 37°C. Incubation time of the strains at 37°C is indicated above the corresponding lanes, and the balance of pre-tRNA^Leu forms in total RNA was isolated by Northern blot. Four major species of tRNA^Leu are the primary transcript, an intermediate containing the intron (“+IVS”), an alternative intermediate containing 5′ and 3′ extensions (“+5,3”), and the mature tRNA^Leu (Lee et al. 1991b). Identities and schematic diagrams of tRNA^Leu species are shown to the left of the blots. Black bars indicate mature tRNA sequences, and lines represent the 5′ leader, the 3′ trailer, and the intervening sequences. The signals of the primary transcript and the intermediate “+5,3” were separately normalized to that of the loading control, SNR190 RNA. The resulting ratios in strain 3HA-Pop1 were set at 1 and were used for comparison with the ratios from other mutant strains. The normalized amounts of primary transcript and “+5,3” intermediate in 3HA-pop1 mutants at 30°C and 37°C (6 h) are listed in Table 2.

FIGURE 4.

Northern blot analysis of precursor and mature RPR1 RNA in representative 3HA-pop1 mutants at 30°C and 37°C. Incubation time of the strains at 37°C is indicated. Precursor and mature RPR1 RNA are detected by Northern blotting, with SNR190 RNA probed as a loading control. Ratio of the mature to precursor RPR1 RNA in each mutant strain at 30°C and 37°C (6 h) is shown in Table 2.

FIGURE 5.

Northern blot analysis of 5.8S rRNA processing in representative 3HA-pop1 mutants at 30°C and 37°C. Incubation time of the strains at 37°C is indicated above the corresponding lanes. Three species of 5.8S rRNA are detected by Northern blotting. These include the long form (“5.8S (L)”), the short form (“5.8 (S)”), and the very long form (“5.8S (VL)”) (Shuai and Warner 1991; Lindahl et al. 1992; Schmitt and Clayton 1993; Chu et al. 1994). The SNR190 RNA is probed as a loading control. Ratio of the short to long forms of 5.8S rRNA in the mutants at 30°C and 37°C (6 h) is summarized in Table 2. Amount of 5.8S (VL) rRNA was normalized to that of SNR190 RNA. The resulting ratio in the control strain 3HA-Pop1 at 37°C was set at 1 and was used for comparison with the ratio from other mutant strains. The normalized amounts of 5.8S (VL) rRNA at 37°C are shown in Table 2.

FIGURE 6.

Northern blot analysis on levels of the NME1 RNA in representative 3HA-pop1 mutants at 30°C and 37°C. Incubation time of the strains at 37°C is indicated. Amount of the NME1 RNA is normalized to SNR190 RNA. The resulting ratio in the control strain 3HA-Pop1 at 30°C was set at 1 and was used for comparison with the ratio from other mutants. The normalized amount of NME1 RNA in each strain at 30°C and 37°C (6 h and 18 h) is listed in Table 2.

FIGURE 7.

Association of the mutated Pop1p with the RPR1 and NME1 RNAs in vivo. (A) Western blotting of 3HA-Pop1p protein in the HA immunoprecipitates. (B) Northern blotting of the NME1 RNA, and precursor and mature RPR1 RNAs copurified with the mutated 3HA-Pop1p. (C) Quantitation of the amounts of 3HA-Pop1p and RNA species in the immunoprecipitates. Signals of the NME1 RNA and the precursor and mature RPR1 RNAs on the Northern blot were divided by the signals of 3HA-Pop1p on the Western blot to account for the difference in the immunoprecipitation efficiency among samples. The resulting ratio in the 3HA-Pop1p control strain was arbitrarily set at 1 and was used for comparison with ratios from the mutant strains.

FIGURE 8.

Summary of the effects of the Pop1p mutations on RNA processing at 37°C. Amino acid sequences of the four conserved regions (CORs) in Pop1p from S. cerevisiae are listed, with positions provided on both sides of the sequences. Effects of the mutations at the highlighted positions on tRNA processing, maturation of the RPR1 RNA, processing of the 5.8S rRNA, and stability of the NME1 RNA are summarized. (+) The mutation causes defects in RNA processing/stability. (−) No obvious defects are detected when the position is mutated. (+/−) The mutated position either impairs RNA processing/stability or does not cause significant defects, depending on the nature of the mutations. Brackets denote that the listed phenotypes of RNA processing are observed when the two indicated positions are mutated at the same time.

FIGURE 9.

Processing of S. cerevisiae pre-tRNA^Tyr in vitro. The cleavage of a pre-tRNA substrate with a 12-nt leader catalyzed by RNase P holoenzyme with either the wild-type (■, at 37°C; □, at 30°C) or Q₂₄₉ mutant (●, at 37°C; ○, at 30°C) Pop1p was determined at either 30°C or 37°C under steady-state conditions. The Michaelis–Menten equation is fit to the data to determine the values for k _cat, K _M, and k _cat/K _M (Table 3).