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 59-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.

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components

“…The size of Pop1, its presence exclusively in the eukaryotic RNases P/MRP Marvin and Esakova and Krasilnikov 2010;Jarrous and Gopalan 2010), the effects of Pop1 mutations on the holoenzymes' assembly (Lygerou et al 1994;Chamberlain et al 1998;Xiao et al 2006), and the apparent spread of the RNase P/MRP RNA regions involved (directly or not) in interactions with Pop1 (Ziehler et al 2001;Houser-Scott et al 2002;Hipp et al 2012;Khanova et al 2012), are all consistent with a potential central role played by Pop1 in the stabilization of the global structure of the RNA components of eukaryotic RNases P/MRP. However, insolubility of individually expressed Pop1 (Ziehler et al 2001;Xiao et al 2006) has previously hindered direct studies of this protein and its interactions with other RNase P/MRP components.…”

“…Like other RNase P/MRP proteins, Pop1 is essential for the viability of the cell (Lygerou et al 1994;Chamberlain et al 1998). Pop1 mutations affect the holoenzyme assembly and result in destabilization of the RNA component in the cell as well as in substrate processing defects in both RNase P and RNase MRP (Lygerou et al 1994;Chamberlain et al 1998;Xiao et al 2006). In humans, certain Pop1 mutations result in a severe skeletal dysplasia (Glazov et al 2011), similar to the dysplasias caused by mutations in the RNase MRP RNA or by defects in RNase MRP RNA promoter region (Ridanpaa et al 2001;Mattijssen et al 2011).…”

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

“…In humans, certain mutations in Pop1, similar to mutations in the RNA component of RNase MRP (Ridanpää et al 2001), result in multisystemic disorders (Glazov et al 2011). Pop1 appears to play multiple roles in the assembly and function of RNases P and MRP (Ziehler et al 2001;Xiao et al 2006).…”

Genome-wide search for yeast RNase P substrates reveals role in maturation of intron-encoded box C/D small nucleolar RNAs

“…Temperaturesensitive RNase P mutations were available in two subunits of yeast RNase P: the unique RNA subunit, Rpr1r (35,36), and the largest protein subunit, Pop1p, that is also a component of RNase MRP (37).…”

“…As part of our screen for possible substrates for RNase P, we performed Northern blot analysis on RNA from RNase P TS strains to see whether some processing intermediates of some small RNAs might accumulate, even though the overall amount of RNA from that transcription unit did not change significantly. Accumulation of pre-tRNAs in these mutants was previously demonstrated (36,37), and we opted to use the TS in the RPR1 RNA subunit because it, unlike Pop1 protein, is unique to RNase P and not found in MRP. Here, 79 additional noncoding RNAs were examined by Northern blot, representing all classes of small nuclear RNAs, small cytoplasmic RNA (SCR1), and box H/ACA and C/D small nucleolar RNAs from independently transcribed, polycistronic, and intron-encoded genes (Table S2).…”