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Journal of Clinical Laboratory Analysis Apr 2021Cell-free DNA (cfDNA) is used in clinical research to identify biomarkers for diagnosis of and follow-up on cancer. Here, we propose a fast and innovative approach using...
BACKGROUND
Cell-free DNA (cfDNA) is used in clinical research to identify biomarkers for diagnosis of and follow-up on cancer. Here, we propose a fast and innovative approach using traditional housekeeping genes as cfDNA targets in a copy number analysis. We focus on the application of highly sensitive technology such as digital PCR (dPCR) to differentiate breast cancer (BC) patients and controls by quantifying regions of PUM1 and RPPH1 (RNase P) in plasma samples.
METHODS
We conducted a case-control study with 82 BC patients and 82 healthy women. cfDNA was isolated from plasma using magnetic beads and quantified by spectrophotometry to estimate total cfDNA. Then, both PUM1 and RPPH1 genes were specifically quantified by dPCR. Data analysis was calibrated using a reference genomic DNA in different concentrations.
RESULTS
We found RNase P and PUM1 values were correlated in the patient group (intraclass correlation coefficient [ICC] = 0.842), but they did not have any correlation in healthy women (ICC = 0.519). In dPCR quantification, PUM1 showed the capacity to distinguish early-stage patients and controls with good specificity (98.67%) and sensitivity (100%). Conversely, RNase P had lower cfDNA levels in triple-negative BC patients than luminal subtypes (p < 0.025 for both), confirming their utility for patient classification.
CONCLUSION
We propose the PUM1 gene as a cfDNA marker for early diagnosis of BC and RNase P as a cfDNA marker related to hormonal status and subtype classification in BC. Further studies with larger sample sizes are warranted.
Topics: Biomarkers, Tumor; Breast Neoplasms; Case-Control Studies; Cell-Free Nucleic Acids; Estrogens; Female; Fluorescence; Humans; Middle Aged; Neoplasm Staging; RNA-Binding Proteins; ROC Curve; Ribonuclease P; Sensitivity and Specificity; Triple Negative Breast Neoplasms
PubMed: 33522650
DOI: 10.1002/jcla.23720 -
Proceedings of the National Academy of... Oct 2017RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium : Without...
RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium : Without an RNA subunit and the smallest of its kind, the 23-kDa polypeptide comprises a metallonuclease domain only. The protein has RNase P activity in vitro and rescued the growth of and strains with inactivations of their more complex and larger endogenous ribonucleoprotein RNase P. Homologs of RNase P (HARP) were identified in many Archaea and some Bacteria, of which all Archaea and most Bacteria also encode an RNA-based RNase P; activity of both RNase P forms from the same bacterium or archaeon could be verified in two selected cases. Bioinformatic analyses suggest that and related likely acquired HARP by horizontal gene transfer from an archaeon.
Topics: Archaea; Bacteria; Gene Transfer, Horizontal; Phylogeny; Ribonuclease P
PubMed: 29073018
DOI: 10.1073/pnas.1707862114 -
The Plant Cell Aug 2021Flowering is the developmental transition from the vegetative to the reproductive phase. FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and...
Flowering is the developmental transition from the vegetative to the reproductive phase. FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), and LEAFY (LFY) are floral integrators. These genes are repressed by several floral repressors including EARLY FLOWERING3 (ELF3), SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO1 (TEM1), and TEM2. Although gibberellin (GA) promotes flowering by activating the floral integrator genes, the exact molecular mechanism remains unclear. DELLAs are negative regulators in GA signaling and act as coactivators of the transcription factor GAI ASSOCIATED FACTOR 1 (GAF1). GAs convert the GAF1 complex from a transcriptional activator to a repressor. Here, we show that GAF1 functions in the GA-dependent flowering pathway by regulating FT and SOC1 expression in Arabidopsis thaliana. We identified four flowering repressors, ELF3, SVP, TEM1, and TEM2, as GAF1-target genes. In response to GAs, GAF1 forms a transcriptional repressor complex and promotes the expression of FT and SOC1 through the repression of four flowering repressor genes, ELF3, SVP, TEM1, and TEM2.
Topics: Arabidopsis; Arabidopsis Proteins; Flowers; Gene Expression Regulation, Plant; Gibberellins; Plants, Genetically Modified; Promoter Regions, Genetic; Protein Binding; Ribonuclease P; Signal Transduction; Transcription Factors
PubMed: 33822231
DOI: 10.1093/plcell/koab102 -
The FEBS Journal Jul 2022Coordination of transcription and processing of RNA is a basic principle in regulation of gene expression in eukaryotes. In the case of mRNA, coordination is primarily... (Review)
Review
Coordination of transcription and processing of RNA is a basic principle in regulation of gene expression in eukaryotes. In the case of mRNA, coordination is primarily founded on a co-transcriptional processing mechanism by which a nascent precursor mRNA undergoes maturation via cleavage and modification by the transcription machinery. A similar mechanism controls the biosynthesis of rRNA. However, the coordination of transcription and processing of tRNA, a rather short transcript, remains unknown. Here, we present a model for high molecular weight initiation complexes of human RNA polymerase III that assemble on tRNA genes and process precursor transcripts to mature forms. These multifunctional initiation complexes may support co-transcriptional processing, such as the removal of the 5' leader of precursor tRNA by RNase P. Based on this model, maturation of tRNA is predetermined prior to transcription initiation.
Topics: Humans; RNA Polymerase III; RNA Precursors; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Transfer; Ribonuclease P; Transcription, Genetic
PubMed: 33929081
DOI: 10.1111/febs.15904 -
The Journal of Biological Chemistry Feb 2020The ribosome and RNase P are cellular ribonucleoprotein complexes that perform peptide bond synthesis and phosphodiester bond cleavage, respectively. Both are ancient... (Review)
Review
The ribosome and RNase P are cellular ribonucleoprotein complexes that perform peptide bond synthesis and phosphodiester bond cleavage, respectively. Both are ancient biological assemblies that were already present in the last universal common ancestor of all life. The large subunit rRNA in the ribosome and the RNA subunit of RNase P are the ribozyme components required for catalysis. Here, we explore the idea that these two large ribozymes may have begun their evolutionary odyssey as an assemblage of RNA "fragments" smaller than the contemporary full-length versions and that they transitioned through distinct stages along a pathway that may also be relevant for the evolution of other non-coding RNAs.
Topics: Evolution, Molecular; Models, Molecular; RNA, Catalytic; RNA, Ribosomal; Ribonuclease P
PubMed: 31953324
DOI: 10.1074/jbc.REV119.009929 -
Methods in Enzymology 2021The ubiquitous ribonucleoprotein (RNP) form of RNase P catalyzes the Mg-dependent cleavage of the 5' leader of precursor-transfer RNAs. The rate and fidelity of the...
The ubiquitous ribonucleoprotein (RNP) form of RNase P catalyzes the Mg-dependent cleavage of the 5' leader of precursor-transfer RNAs. The rate and fidelity of the single catalytic RNA subunit in the RNase P RNP is significantly enhanced by association with protein cofactors. While the bacterial RNP exhibits robust activity at near-physiological Mg concentrations with a single essential protein cofactor, archaeal and eukaryotic RNase P are dependent on up to 5 and 10 protein subunits, respectively. Archaeal RNase P-whose proteins share eukaryotic homologs-is an experimentally tractable model for dissecting in a large RNP the roles of multiple proteins that aid an RNA catalyst. We describe protocols to assemble RNase P from Methanococcus maripaludis, a methanogenic archaeon. We present strategies for tag-less purification of four of the five proteins (the tag from the fifth is removed post-purification), an approach that helps reconstitute the RNase P RNP with near-native constituents. We demonstrate the value of native mass spectrometry (MS) in establishing the accurate masses (including native oligomers and modifications) of all six subunits in M. maripaludis RNase P, and the merits of mass photometry (MP) as a complement to native MS for characterizing the oligomeric state of protein complexes. We showcase the value of native MS and MP in revealing time-dependent modifications (e.g., oxidation) and aggregation of protein subunits, thereby providing insights into the decreased function of RNase P assembled with aged preparations of recombinant subunits. Our protocols and cautionary findings are applicable to studies of other cellular RNPs.
Topics: Archaea; Archaeal Proteins; RNA; RNA Precursors; RNA, Catalytic; Ribonuclease P
PubMed: 34752299
DOI: 10.1016/bs.mie.2021.07.006 -
The Journal of Biological Chemistry Dec 2022The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a...
The first step in transfer RNA (tRNA) maturation is the cleavage of the 5' end of precursor tRNA (pre-tRNA) catalyzed by ribonuclease P (RNase P). RNase P is either a ribonucleoprotein complex with a catalytic RNA subunit or a protein-only RNase P (PRORP). In most land plants, algae, and Euglenozoa, PRORP is a single-subunit enzyme. There are currently no inhibitors of PRORP for use as tools to study the biological function of this enzyme. Therefore, we screened for compounds that inhibit the activity of a model PRORP from A. thaliana organelles (PRORP1) using a high throughput fluorescence polarization cleavage assay. Two compounds, gambogic acid and juglone (5-hydroxy-1,4-naphthalenedione) that inhibit PRORP1 in the 1 μM range were identified and analyzed. We found these compounds similarly inhibit human mtRNase P, a multisubunit protein enzyme and are 50-fold less potent against bacterial RNA-dependent RNase P. Our biochemical measurements indicate that gambogic acid is a rapid-binding, uncompetitive inhibitor targeting the PRORP1-substrate complex, while juglone acts as a time-dependent PRORP1 inhibitor. Additionally, X-ray crystal structures of PRORP1 in complex with juglone demonstrate the formation of a covalent complex with cysteine side chains on the surface of the protein. Finally, we propose a model consistent with the kinetic data that involves juglone binding to PRORP1 rapidly to form an inactive enzyme-inhibitor complex and then undergoing a slow step to form an inactive covalent adduct with PRORP1. These inhibitors have the potential to be developed into tools to probe PRORP structure and function relationships.
Topics: Humans; Arabidopsis; Arabidopsis Proteins; Naphthoquinones; Ribonuclease P; RNA Precursors; RNA, Transfer
PubMed: 36370850
DOI: 10.1016/j.jbc.2022.102683 -
Antimicrobial Agents and Chemotherapy Jul 2021RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a...
RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies have reported small-molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis, and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 (and its derivatives), iriginol hexaacetate, and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg ions. These findings were deduced from solubility analyses by microscopy and high-performance liquid chromatography (HPLC), RnpA-inhibitor co-pulldown experiments, detergent addition, and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported nonspecific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of "false positives" that are also termed an-ssay terference compound (PAINS).
Topics: Bacillus subtilis; High-Throughput Screening Assays; Humans; RNA, Bacterial; Ribonuclease P; Staphylococcal Infections; Staphylococcus aureus
PubMed: 33972249
DOI: 10.1128/AAC.00300-21 -
RNA (New York, N.Y.) Mar 2023The seminal discovery of ribonuclease P (RNase P) and its catalytic RNA by Sidney Altman has not only revolutionized our understanding of life, but also opened new... (Review)
Review
The seminal discovery of ribonuclease P (RNase P) and its catalytic RNA by Sidney Altman has not only revolutionized our understanding of life, but also opened new fields for scientific exploration and investigation. This review focuses on human RNase P and its use as a gene-targeting tool, two topics initiated in Altman's laboratory. We outline early works on human RNase P as a tRNA processing enzyme and comment on its expanding nonconventional functions in molecular networks of transcription, chromatin remodeling, homology-directed repair, and innate immunity. The important implications and insights from these discoveries on the potential use of RNase P as a gene-targeting tool are presented. This multifunctionality calls to a modified structure-function partitioning of domains in human RNase P, as well as its relative ribonucleoprotein, RNase MRP. The role of these two catalysts in innate immunity is of particular interest in molecular evolution, as this dynamic molecular network could have originated and evolved from primordial enzymes and sensors of RNA, including predecessors of these two ribonucleoproteins.
Topics: Humans; Ribonuclease P; RNA; RNA Processing, Post-Transcriptional; RNA, Catalytic
PubMed: 36549864
DOI: 10.1261/rna.079475.122 -
The Journal of Biological Chemistry Nov 2023tRNAs are typically transcribed with extended 5' and 3' ends that must be removed before they attain their active form. One of the first steps of tRNA processing in...
tRNAs are typically transcribed with extended 5' and 3' ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5' leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered class of RNase P enzymes, Homologs of Aquifex RNase P (HARPs). In contrast to other RNase Ps, HARPs consist only of a metallonuclease domain and lack the canonical substrate recognition domain essential in other classes of proteinaceous RNase P. We determined the cryo-EM structure of Aquifex aeolicus HARP (Aq880) and two crystal structures of Hydrogenobacter thermophilus HARP (Hth1307) to reveal that both enzymes form large ring-like assemblies: a dodecamer in Aq880 and a tetradecamer in Hth1307. In both oligomers, the enzyme active site is 42 Å away from a positively charged helical region, as seen in other protein-only RNase P enzymes, which likely serves to recognize and bind the elbow region of the pre-tRNA substrate. In addition, we use native mass spectrometry to confirm and characterize the previously unreported tetradecamer state. Notably, we find that multiple oligomeric states of Hth1307 are able to cleave pre-tRNAs. Furthermore, our single-turnover kinetic studies indicate that Hth1307 cleaves pre-tRNAs from multiple species with a preference for native substrates. These data provide a closer look at the nuanced similarities and differences in tRNA processing across disparate classes of RNase P.
Topics: Ribonuclease P; RNA, Bacterial; Kinetics; Nucleic Acid Conformation; RNA, Transfer; Bacteria; RNA Precursors
PubMed: 37806495
DOI: 10.1016/j.jbc.2023.105327