Probing the structure of Saccharomyces cerevisiae RNase MRP

2005 ◽  
Vol 33 (3) ◽  
pp. 479-481 ◽  
Author(s):  
S.C. Walker ◽  
T.V. Aspinall ◽  
J.M.B. Gordon ◽  
J.M. Avis
Keyword(s):  
Rna Structure ◽  
Rna Stability ◽  
Rnase P ◽  
In Vivo Studies ◽  
Mitochondrial Rna ◽  
Protein Subunits ◽  
Equivalent Position ◽  
Rnase Mrp ◽  
Alternative Structure

In yeast, RNase MRP (mitochondrial RNA processing), a ribonucleoprotein precursor rRNA processing enzyme, possesses one putatively catalytic RNA and ten protein subunits and is highly related to RNase P. Structural analysis of the MRP RNA provides data that closely match a previous secondary-structure model derived from phylogenetic analysis, with the exception of an additional stem. This stem occupies an equivalent position to the P7 stem of RNase P RNA and its inclusion confers on MRP RNA a greater similarity to the core P RNA structure. In vivo studies indicate that the P7-like stem can form, but is not a part of, the active enzyme structure. Stem formation would increase RNA stability in the absence of proteins and our alternative structure may be a valid intermediate species in RNase MRP assembly. Further ongoing studies of this enzyme reveal an extensive network of interactions between subunits and a probable central role for the Pop1, Pop4 and Pop7 subunits.

scholarly journals Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

2018 ◽  
Author(s):  
Agnes Karasik ◽  
Carol A. Fierke ◽  
Markos Koutmos
Keyword(s):  
Mitochondrial Diseases ◽  
Protein S ◽  
Rnase P ◽  
Ribonuclease P ◽  
Mitochondrial Rna ◽  
P Protein ◽  
Adenosyl Methionine ◽  
Methylation Activity

ABSTRACTHuman mitochondrial ribonuclease P (mtRNase P) is an essential three protein complex that catalyzes the 5’ end maturation of mitochondrial precursor tRNAs (pre-tRNAs). MRPP3 (Mitochondrial RNase P Protein 3), a protein-only RNase P (PRORP), is the nuclease component of the mtRNase P complex and requires a two-protein S-adenosyl methionine (SAM)-dependent methyltransferase MRPP1/2 sub-complex to function. Dysfunction of mtRNase P is linked to several human mitochondrial diseases, such as mitochondrial myopathies. Despite its central role in mitochondrial RNA processing, little is known about how the protein subunits of mtRNase P function synergistically. Here we use purified mtRNase P to demonstrate that mtRNase P recognizes, cleaves, and methylates some, but not all, mitochondrial pre-tRNAs in vitro. Additionally, mtRNase P does not process all mitochondrial pre-tRNAs uniformly, suggesting the possibility that some pre-tRNAs require additional factors to be cleaved in vivo. Consistent with this, we found that addition of the MRPP1 co-factor SAM enhances the ability of mtRNase P to bind and cleave some mitochondrial pre-tRNAs. Furthermore, the presence of MRPP3 can enhance the methylation activity of MRPP1/2. Taken together, our data demonstrate that the subunits of mtRNase P work together to efficiently recognize, process and methylate human mitochondrial pre-tRNAs.

scholarly journals Efficient site-specific cleavage by RNase MRP requires interaction with two evolutionarily conserved mitochondrial RNA sequences.

1990 ◽  
Vol 10 (5) ◽  
pp. 2191-2201 ◽  
Author(s):  
J L Bennett ◽  
D A Clayton
Keyword(s):  
Sequence Homology ◽  
Mammalian Species ◽  
Saturation Mutagenesis ◽  
Mitochondrial Rna ◽  
Rna Sequences ◽  
Site Specific ◽  
Conserved Sequence ◽  
Sequence Block ◽  
Rnase Mrp

RNase MRP is a site-specific endonuclease that processes primer mitochondrial RNA from the leading-strand origin of mitochondrial DNA replication. Using deletional analysis and saturation mutagenesis, we have determined the substrate requirements for cleavage by mouse mitochondrial RNase MRP. Two regions of sequence homology among vertebrate mitochondrial RNA primers, conserved sequence blocks II and III, were found to be critical for both efficient and accurate cleavage; a third region of sequence homology, conserved sequence block I, was dispensable. Analysis of insertion and deletion mutations within conserved sequence block II demonstrated that the specificity of RNase MRP accommodates the natural sequence heterogeneity of conserved sequence block II in vivo. Heterologous assays with human RNase MRP and mutated mouse mitochondrial RNA substrates indicated that sequences essential for substrate recognition are conserved between mammalian species.

scholarly journals Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association

2001 ◽  
Vol 12 (11) ◽  
pp. 3680-3689 ◽  
Author(s):  
Hans van Eenennaam ◽  
Annemarie van der Heijden ◽  
Rolf J. R. J. Janssen ◽  
Walther J. van Venrooij ◽  
Ger J. M. Pruijn
Keyword(s):  
Protein Composition ◽  
Rnase P ◽  
Nucleolar Localization ◽  
Rrna Processing ◽  
Protein Subunits ◽  
Trna Processing ◽  
Rnase Mrp ◽  
Human Rnase ◽  
The Difference ◽  
Active Transport Process

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.

scholarly journals Biogenesis of RNase P RNA from an intron requires co-assembly with cognate protein subunits

2019 ◽  
Vol 47 (16) ◽  
pp. 8746-8754 ◽  
Author(s):  
Geeta Palsule ◽  
Venkat Gopalan ◽  
Amanda Simcox
Keyword(s):  
Noncoding Rnas ◽  
Rnase P ◽  
Pol Ii ◽  
Protein Subunits ◽  
Mature Form ◽  
Rna Exosome ◽  
P Proteins ◽  
Pol Iii ◽  
Cognate Protein

Abstract RNase P RNA (RPR), the catalytic subunit of the essential RNase P ribonucleoprotein, removes the 5′ leader from precursor tRNAs. The ancestral eukaryotic RPR is a Pol III transcript generated with mature termini. In the branch of the arthropod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II transcript and requires processing from intron sequences for maturation. We demonstrate here that the Drosophila intronic-RPR precursor is trimmed to the mature form by the ubiquitous nuclease Rat1/Xrn2 (5′) and the RNA exosome (3′). Processing is regulated by a subset of RNase P proteins (Rpps) that protects the nascent RPR from degradation, the typical fate of excised introns. Our results indicate that the biogenesis of RPR in vivo entails interaction of Rpps with the nascent RNA to form the RNase P holoenzyme and suggests that a new pathway arose in arthropods by coopting ancient mechanisms common to processing of other noncoding RNAs.

scholarly journals The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3′-UTRs of yeast mitochondrial mRNAs

2021 ◽  
Vol 118 (15) ◽  
pp. e2009329118
Author(s):  
Hauke S. Hillen ◽  
Dmitriy A. Markov ◽  
Ireneusz D. Wojtas ◽  
Katharina B. Hofmann ◽  
Michael Lidschreiber ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Rna Stability ◽  
Mitochondrial Rna ◽  
Pentatricopeptide Repeat ◽  
Sequence Element ◽  
Posttranscriptional Gene Regulation ◽  
The Family ◽  
Ppr Proteins

Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5′ capping and 3′ polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3′-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3′-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.

Efficient site-specific cleavage by RNase MRP requires interaction with two evolutionarily conserved mitochondrial RNA sequences

1990 ◽  
Vol 10 (5) ◽  
pp. 2191-2201
Author(s):  
J L Bennett ◽  
D A Clayton
Keyword(s):  
Sequence Homology ◽  
Mammalian Species ◽  
Saturation Mutagenesis ◽  
Mitochondrial Rna ◽  
Rna Sequences ◽  
Site Specific ◽  
Conserved Sequence ◽  
Sequence Block ◽  
Rnase Mrp

RNase MRP is a site-specific endonuclease that processes primer mitochondrial RNA from the leading-strand origin of mitochondrial DNA replication. Using deletional analysis and saturation mutagenesis, we have determined the substrate requirements for cleavage by mouse mitochondrial RNase MRP. Two regions of sequence homology among vertebrate mitochondrial RNA primers, conserved sequence blocks II and III, were found to be critical for both efficient and accurate cleavage; a third region of sequence homology, conserved sequence block I, was dispensable. Analysis of insertion and deletion mutations within conserved sequence block II demonstrated that the specificity of RNase MRP accommodates the natural sequence heterogeneity of conserved sequence block II in vivo. Heterologous assays with human RNase MRP and mutated mouse mitochondrial RNA substrates indicated that sequences essential for substrate recognition are conserved between mammalian species.

scholarly journals The Dynamic Network of RNP RNase P Subunits

2021 ◽  
Vol 22 (19) ◽  
pp. 10307
Author(s):  
Athanasios-Nasir Shaukat ◽  
Eleni G. Kaliatsi ◽  
Ilias Skeparnias ◽  
Constantinos Stathopoulos
Keyword(s):  
Dynamic Network ◽  
Rnase P ◽  
Ribonuclease P ◽  
Protein Subunit ◽  
Protein Subunits ◽  
Phosphodiester Bond ◽  
Cellular Processes ◽  
Protein Partners

Ribonuclease P (RNase P) is an important ribonucleoprotein (RNP), responsible for the maturation of the 5′ end of precursor tRNAs (pre-tRNAs). In all organisms, the cleavage activity of a single phosphodiester bond adjacent to the first nucleotide of the acceptor stem is indispensable for cell viability and lies within an essential catalytic RNA subunit. Although RNase P is a ribozyme, its kinetic efficiency in vivo, as well as its structural variability and complexity throughout evolution, requires the presence of one protein subunit in bacteria to several protein partners in archaea and eukaryotes. Moreover, the existence of protein-only RNase P (PRORP) enzymes in several organisms and organelles suggests a more complex evolutionary timeline than previously thought. Recent detailed structures of bacterial, archaeal, human and mitochondrial RNase P complexes suggest that, although apparently dissimilar enzymes, they all recognize pre-tRNAs through conserved interactions. Interestingly, individual protein subunits of the human nuclear and mitochondrial holoenzymes have additional functions and contribute to a dynamic network of elaborate interactions and cellular processes. Herein, we summarize the role of each RNase P subunit with a focus on the human nuclear RNP and its putative role in flawless gene expression in light of recent structural studies.

scholarly journals RNase MRP Cleaves the CLB2 mRNA To Promote Cell Cycle Progression: Novel Method of mRNA Degradation

2004 ◽  
Vol 24 (3) ◽  
pp. 945-953 ◽  
Author(s):  
Tina Gill ◽  
Ti Cai ◽  
Jason Aulds ◽  
Sara Wierzbicki ◽  
Mark E. Schmitt
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mrna Degradation ◽  
Molecular Defect ◽  
Mitochondrial Rna ◽  
Mrna Levels ◽  
Vitro Assay ◽  
Rnase Mrp

ABSTRACT RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5′ untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5′→3′ exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5′-UTR to allow rapid 5′ to 3′ degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.

Abstract 202: Hydrogen Peroxide Increases Osteopontin Expression Through Activation of Transcriptional and Translational Pathways

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Alicia N Lyle ◽  
Ebony W Remus ◽  
Aaron E Fan ◽  
W Robert Taylor
Keyword(s):  
Transcriptional Regulation ◽  
Translational Regulation ◽  
Rna Stability ◽  
Rna Degradation ◽  
Initiation Factor ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Time Courses

The occlusion of blood vessels in the setting of cardiovascular disease leads to ischemia, initiating processes that promote neovascularization to restore blood flow and preserve tissue function. Our in vivo studies show that Osteopontin (OPN) is a critical mediator of post-ischemic neovascularization and that ischemia-induced increases in OPN expression are H 2 O 2 -dependent. However, the mechanisms by which H 2 O 2 increases OPN expression are poorly defined. To determine if H 2 O 2 mediates transcriptional, post-transcriptional, and/or translational regulation of OPN expression in vitro, we used rat aortic smooth muscle cells as an in vitro system and stimulated with H 2 O 2 . Dose response studies showed OPN expression increased with 50 μM H 2 O 2 (51.9%±2.2, p<0.05). Using 50 μM H 2 O 2 , we performed time courses and measured OPN mRNA by qRT-PCR and protein by Western blot. OPN mRNA levels significantly increased in response to H 2 O 2 at 8 (70.4%±5.7, p<0.05) and 18 hours (120.2%±5.2, p<0.005). Interestingly, the increases in OPN protein expression in response to H 2 O 2 occurred in an unusual bi-phasic pattern, with significant increases at 6 (96.9%±1.5, p<0.001) and 18 hours (234.0%±3.6, p<0.001), with a return to baseline in between. An increase in OPN mRNA preceded the increase in OPN protein at 18 hours, suggesting transcriptional regulation; however, the acute increase in OPN at 6 hours was not preceded by increased mRNA, suggesting multiple mechanisms of OPN regulation by H 2 O 2 . To determine if the increase in OPN at 6 hours is due to increased mRNA stability or translation, we performed an RNA stability assay. H 2 O 2 stimulation did not alter OPN stability or the rate of OPN RNA degradation, leading us to conclude the increase in OPN expression at 6 hours is due to increased translation. Further studies reveal H 2 O 2 -mediated increases in phosphorylation of 4E-BP1 at the redox-sensitive Ser65 site (89.4%±6.1, p<0.05), allowing for the subsequent release of eukaryotic initiation factor eIF4E and increased phosphorylation at Ser209 (139.2%±3.9, p<0.05), resulting in increased OPN translation. In conclusion, H 2 O 2 enhances OPN expression through acute increases in translation, while long-term increases in OPN occur through increased transcriptional regulation.

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