Global Identification of New Substrates for the Yeast Endoribonuclease, RNase Mitochondrial RNA Processing (MRP).
ABSTRACT RNase MRP is an essential, evolutionarily conserved endoribonuclease composed of 10 different protein subunits and a single RNA. RNase MRP has established roles in multiple pathways including: ribosome biogenesis, cell cycle regulation, and mitochondrial DNA replication. Although each of these functions is important to cell growth additional functions may exist given the essential nature of the complex. In order to identify novel RNase MRP substrates, we have utilized RIP-chip (RNA Immuno-Precipitation and microarray chip analysis) to identify RNA that physically associates with RNase MRP. We identified several new potential substrates for RNase MRP including a cell-cycle regulated transcript, CTS1, the yeast homolog of the mammalian p27(Kip1), SIC1, and the U2 RNA component of the spliceosome. In addition, we found RNase MRP to be involved in the regulation of the TY1 transposon RNA. These results reinforce and broaden the role of RNase MRP in cell cycle regulation and help to identify new roles of this endoribonuclease.
[Show abstract] [Hide abstract]
ABSTRACT: Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.Nucleic Acids Research 05/2013; 41(14). DOI:10.1093/nar/gkt432 · 8.81 Impact FactorThis article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.
SourceAvailable from: Kouji Hirota[Show abstract] [Hide abstract]
ABSTRACT: Ribonuclease mitochondrial RNA processing (RNase MRP) is a multifunctional ribonucleoprotein (RNP) complex that is involved in the maturation of various types of RNA including ribosomal RNA. RNase MRP consists of a potential catalytic RNA and several protein components, all of which are required for cell viability. We show here that the temperature-sensitive mutant of rmp1, the gene for a unique protein component of RNase MRP, accumulates the dimeric tRNA precursor, pre-tRNASer-Met. To examine whether RNase MRP mediates tRNA maturation, we purified the RNase MRP holoenzyme from the fission yeast Schizosaccharomyces pombe and found that the enzyme directly and selectively cleaves pre-tRNASer-Met, suggesting that RNase MRP participates in the maturation of specific tRNA in vivo. In addition, mass spectrometry-based ribonucleoproteomic analysis demonstrated that this RNase MRP consists of one RNA molecule and 11 protein components, including a previously unknown component Rpl701. Notably, limited nucleolysis of RNase MRP generated an active catalytic core consisting of partial mrp1 RNA fragments, which constitute "Domain 1" in the secondary structure of RNase MRP, and 8 proteins. Thus, the present study provides new insight into the structure and function of RNase MRP.PLoS ONE 11/2014; 9(11):e112488. DOI:10.1371/journal.pone.0112488 · 3.53 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of precursor-tRNAs (pre-tRNA) and is conserved in all domains of life. However, the composition of RNase P varies from bacteria to archaea and eukarya, making RNase P one of the most diverse enzymes characterized. Most known RNase P enzymes contain a large catalytic RNA subunit that associates with one to 10 proteins. Recently, a protein-only form of RNase P was discovered in mitochondria and chloroplasts of many higher eukaryotes. This proteinaceous RNase P (PRORP) represents a new class of metallonucleases. Here we discuss our recent crystal structure of PRORP1 from Arabidopsis thaliana and speculate on the reasons for the replacement of catalytic RNA by a protein catalyst. We conclude, based on an analysis of the catalytic efficiencies of ribonucleoprotein (RNP) and PRORP enzymes, that the need for greater catalytic efficiency is most likely not the driving force behind the replacement of the RNA with a protein catalyst. The emergence of a protein-based RNase P more likely reflects the increasing complexity of the biological system, including difficulties in importation into organelles and vulnerability of organellar RNAs to cleavage.RNA biology 04/2013; 10(6). DOI:10.4161/rna.24513 · 5.38 Impact Factor