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.

In Vitro and in Vivo Processing of Cyanelle tmRNA by RNase P

2001 ◽  
Vol 382 (10) ◽  
pp. 1421-1429 ◽  
Author(s):  
O. Gimple ◽  
A. Schön
Keyword(s):  
Rnase P ◽  
Ribonuclease P ◽  
Structure Model ◽  
Cyanophora Paradoxa ◽  
Secondary Structure Model ◽  
Precursor Molecule ◽  
Its Gene ◽  
Multicellular Organisms

Abstract Ribonuclease P, the ubiquitous endonuclease required for generating mature tRNA 5 ends, is a ribonucleoprotein in most organisms and organelles, with the exception of mitochondria and chloroplasts of multicellular organisms. The cyanelle of the primitive alga Cyanophora paradoxa is the only photosynthetic organelle where the ribonucleoprotein nature of this enzyme has been functionally proven. tmRNA is another highly structured RNA: it can be aminoacylated with alanine, which is then incorporated into a tag peptide encoded on the same RNA molecule. This dualfunction RNA has been found in bacteria, and its gene is also present in mitochondria and plastids from primitive organisms. Since nothing is known about the expression of this RNA in organelles, we have performed processing studies and determined the promoter of cyanelle pretmRNA. This RNA is transcribed as a precursor molecule in vivo. Synthetic transcripts of cyanelle pretmRNA, including or lacking the mature 3 CCAend, are efficiently and correctly processed in vitro by bacterial RNase P ribo and holoenzymes and by the homologous cyanelle RNase P. In addition to these experimental data, we propose a novel secondary structure model for this organellar tmRNA, which renders it more similar to its bacterial counterpart.

scholarly journals Kinetic mechanism of human mitochondrial RNase P

2019 ◽  
Author(s):  
Xin Liu ◽  
Nancy Wu ◽  
Aranganathan Shanmuganathan ◽  
Bradley P. Klemm ◽  
Michael J. Howard ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rnase P ◽  
Kinetic Mechanism ◽  
Ribonuclease P ◽  
Substrate Affinity ◽  
P Protein ◽  
Binding Data ◽  
Catalytically Active

ABSTRACTA first step in processing mitochondrial precursor tRNA (pre-tRNA) is cleavage of the 5’ leader catalyzed by ribonuclease P (RNase P). Human mitochondrial RNase P (mtRNase P) is composed of three protein subunits: mitochondrial RNase P protein (MRPP) 1, 2 and 3. Even though MRPP3 is the metallonuclease subunit responsible for catalysis, cleavage is observed only in the presence of the MRPP1/2 subcomplex. To understand the functional role of MRPP1/2, we reconstituted human mitochondrial RNase P in vitro and performed kinetic and thermodynamic analyses. MRPP1/2 significantly enhances both the catalytic activity and the apparent substrate affinity of mtRNase P. Additionally, pull-down and binding data demonstrate synergy between binding pre-tRNA and formation of a catalytically active MRPP1/2/3 complex. These data suggest that conformational changes in the MRPP1/2-pre-tRNA complex lead to protein-protein or protein-RNA interactions that increase both MRPP3 recognition and cleavage efficiency. This work presents the first kinetic model for human mtRNase P, providing a fundamental framework for the function of MRPP1/2 for recognition and processing of pre-tRNA.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

scholarly journals Towards plant resistance to viruses using protein-only RNase P

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anthony Gobert ◽  
Yifat Quan ◽  
Mathilde Arrivé ◽  
Florent Waltz ◽  
Nathalie Da Silva ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Virus Replication ◽  
Yield Loss ◽  
Rnase P ◽  
Plant Viruses ◽  
Yellow Mosaic Virus ◽  
Resistance To Viruses ◽  
Target Plant

AbstractPlant viruses cause massive crop yield loss worldwide. Most plant viruses are RNA viruses, many of which contain a functional tRNA-like structure. RNase P has the enzymatic activity to catalyze the 5′ maturation of precursor tRNAs. It is also able to cleave tRNA-like structures. However, RNase P enzymes only accumulate in the nucleus, mitochondria, and chloroplasts rather than cytosol where virus replication takes place. Here, we report a biotechnology strategy based on the re-localization of plant protein-only RNase P to the cytosol (CytoRP) to target plant viruses tRNA-like structures and thus hamper virus replication. We demonstrate the cytosol localization of protein-only RNase P in Arabidopsis protoplasts. In addition, we provide in vitro evidences for CytoRP to cleave turnip yellow mosaic virus and oilseed rape mosaic virus. However, we observe varied in vivo results. The possible reasons have been discussed. Overall, the results provided here show the potential of using CytoRP for combating some plant viral diseases.

Protein synthesis is required for rapid degradation of tubulin mRNA and other deflagellation-induced RNAs in Chlamydomonas reinhardi

1986 ◽  
Vol 6 (1) ◽  
pp. 54-61
Author(s):  
E J Baker ◽  
L R Keller ◽  
J A Schloss ◽  
J L Rosenbaum
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Control ◽  
Protein S ◽  
Protein Synthesis Inhibitors ◽  
Control Factors ◽  
Flagellar Proteins ◽  
The Stability ◽  
And Control

After flagellar detachment in Chlamydomonas reinhardi, there is a rapid synthesis and accumulation of mRNAs for tubulin and other flagellar proteins. Maximum levels of these mRNAs (flagellar RNAs) are reached within 1 h after deflagellation, after which they are rapidly degraded to their predeflagellation levels. The degradation of alpha- and beta-tubulin RNAs was shown to be due to the shortening of their half-lives after accumulation (Baker et al., J. Cell Biol. 99:2074-2081, 1984). Deflagellation in the presence of protein synthesis inhibitors results in the accumulation of tubulin and other flagellar mRNAs by kinetics similar to those of controls. However, unlike controls, in which the accumulated mRNAs are rapidly degraded, these mRNAs are stabilized in cycloheximide. The stabilization by cycloheximide is specific for the flagellar mRNAs accumulated after deflagellation, since there is no change in the levels of flagellar mRNAs in nondeflagellated (uninduced) cells in the presence of cycloheximide. The kinetics of flagellar mRNA synthesis after deflagellation are shown to be the same in cycloheximide-treated and control cells by in vivo labeling and in vitro nuclear runoff experiments. These results show that protein synthesis is not required for the induced synthesis of flagellar mRNAs, and that all necessary transcriptional control factors are present in the cell before deflagellation, but that protein synthesis is required for the accelerated degradation of the accumulated flagellar mRNAs. Since cycloheximide prevents the induced synthesis and accumulation of flagellar proteins, it is possible that the product(s) of protein synthesis required for the accelerated decay of these mRNAs is a flagellar protein(s). The possibility that one or more flagellar proteins autoregulate the stability of the flagellar mRNAs is discussed.

Synthetic Riboswitches for the Analysis of tRNA Processing by eukaryotic RNase P Enzymes

RNA ◽  
2022 ◽  
pp. rna.078814.121
Author(s):  
Anna Ender ◽  
Nadine Grafl ◽  
Tim Kolberg ◽  
Sven Findeiss ◽  
Peter F. Stadler ◽  
...  
Keyword(s):  
Homo Sapiens ◽  
Rnase P ◽  
Single Stranded Region ◽  
Domains Of Life ◽  
The Individual ◽  
The Impact ◽  
Individual Enzyme ◽  
Trna Precursor

Removal of the 5' leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5' leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes – two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

scholarly journals A Nonerythroid Isoform of Protein 4.1R Interacts with Components of the Contractile Apparatus in Skeletal Myofibers

2000 ◽  
Vol 11 (11) ◽  
pp. 3805-3817 ◽  
Author(s):  
Aikaterini Kontrogianni-Konstantopoulos ◽  
Shu-Ching Huang ◽  
Edward J. Benz
Keyword(s):  
Skeletal Muscle ◽  
Protein S ◽  
Binding Assays ◽  
Adult Skeletal Muscle ◽  
Exon 2 ◽  
Alternatively Spliced ◽  
Translation Initiation Codon ◽  
Protein 4.1R

The ∼80-kDa erythroid 4.1R protein is a major component of the erythrocyte cytoskeleton, where it links transmembrane proteins to the underlying spectrin/actin complexes. A diverse collection of 4.1R isoforms has been described in nonerythroid cells, ranging from ∼30 to ∼210 kDa. In the current study, we identified the number and primary structure of 4.1R isoforms expressed in adult skeletal muscle and characterized the localization patterns of 4.1R message and protein. Skeletal muscle 4.1R appears to originate solely from the upstream translation initiation codon (AUG-1) residing in exon 2′. Combinations of alternatively spliced downstream exons generate an array of distinct 4.1R spliceoforms. Two major isoform classes of ∼105/110 and ∼135 kDa are present in muscle homogenates. 4.1R transcripts are distributed in highly ordered signal stripes, whereas 4.1R protein(s) decorate the sarcoplasm in transverse striations that are in register with A-bands. An ∼105/110-kDa 4.1R isoform appears to occur in vivo in a supramolecular complex with major sarcomeric proteins, including myosin, α-actin, and α-tropomyosin. In vitro binding assays showed that 4.1R may interact directly with the aforementioned contractile proteins through its 10-kDa domain. All of these observations suggest a topological model whereby 4.1R may play a scaffolding role by anchoring the actomyosin myofilaments and possibly modulating their displacements during contraction/relaxation.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies

2012 ◽  
Vol 107 (03) ◽  
pp. 468-476 ◽  
Author(s):  
Ilze Dienava-Verdoold ◽  
Marina R. Marchetti ◽  
Liane C. J. te Boome ◽  
Laura Russo ◽  
Anna Falanga ◽  
...  
Keyword(s):  
Protein C ◽  
Normal Values ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Intact Protein ◽  
The Impact ◽  
Independent Protein

SummaryThe natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

scholarly journals A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor

2019 ◽  
Vol 47 (12) ◽  
pp. 6425-6438 ◽  
Author(s):  
Ezequiel-Alejandro Madrigal-Carrillo ◽  
Carlos-Alejandro Díaz-Tufinio ◽  
Hugo-Aníbal Santamaría-Suárez ◽  
Marcelino Arciniega ◽  
Alfredo Torres-Larios
Keyword(s):  
Rna Processing ◽  
Rnase P ◽  
Ribonuclease P ◽  
Binding Assays ◽  
Antibiotic Development ◽  
Processing Enzymes ◽  
P Protein ◽  
Molecule Inhibitor ◽  
Screening Platform ◽  
Dynamics Simulations

AbstractRibonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5′ tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5′ leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

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