Rational Design of an Orthogonal Pair of Bimolecular RNase P Ribozymes through Heterologous Assembly of Their Modular Domains

The modular structural domains of multidomain RNA enzymes can often be dissected into separate domain RNAs and their noncovalent assembly can often reconstitute active enzymes. These properties are important to understand their basic characteristics and are useful for their application to RNA-based nanostructures. Bimolecular forms of bacterial RNase P ribozymes consisting of S-domain and C-domain RNAs are attractive as platforms for catalytic RNA nanostructures, but their S-domain/C-domain assembly was not optimized for this purpose. Through analysis and engineering of bimolecular forms of the two bacterial RNase P ribozymes, we constructed a chimeric ribozyme with improved catalytic ability and S-domain/C-domain assembly and developed a pair of bimolecular RNase P ribozymes the assembly of which was considerably orthogonal to each other.

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Hyperprocessing of tRNA by the catalytic RNA of RNase P. Cleavage of a natural tRNA within the mature tRNA sequence and evidence for an altered conformation of the substrate tRNA.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)49792-5 ◽

1992 ◽

Vol 267 (17) ◽

pp. 11972-11976

Author(s):

Y Kikuchi ◽

N Sasaki

Keyword(s):

Rnase P ◽

Catalytic Rna ◽

Trna Sequence

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Cleavage of tRNA within the mature tRNA sequence by the catalytic RNA of RNase P: implication for the formation of the primer tRNA fragment for reverse transcription in copia retrovirus-like particles.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.20.8105 ◽

1990 ◽

Vol 87 (20) ◽

pp. 8105-8109 ◽

Cited By ~ 14

Author(s):

Y. Kikuchi ◽

N. Sasaki ◽

Y. Ando-Yamagami

Keyword(s):

Reverse Transcription ◽

Rnase P ◽

Catalytic Rna ◽

Trna Sequence

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Effective inhibition in animals of viral pathogenesis by a ribozyme derived from RNase P catalytic RNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0804922105 ◽

2008 ◽

Vol 105 (31) ◽

pp. 10919-10924 ◽

Cited By ~ 15

Author(s):

Y. Bai ◽

P. Trang ◽

H. Li ◽

K. Kim ◽

T. Zhou ◽

...

Keyword(s):

Rnase P ◽

Viral Pathogenesis ◽

Catalytic Rna ◽

Effective Inhibition

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Identifying Structural Domains and Conserved Regions in the Long Non-Coding RNA lncTCF7

International Journal of Molecular Sciences ◽

10.3390/ijms20194770 ◽

2019 ◽

Vol 20 (19) ◽

pp. 4770 ◽

Cited By ~ 3

Author(s):

Michael C. Owens ◽

Sean C. Clark ◽

Allison Yankey ◽

Srinivas Somarowthu

Keyword(s):

Structure Function ◽

Cancer Biology ◽

Structural Model ◽

Structural Domains ◽

Conserved Regions ◽

Non Coding Rna ◽

Function Relationship ◽

Modular Domains ◽

Relationship Of ◽

Long Non Coding Rna

Long non-coding RNA (lncRNA) biology is a rapidly growing area of study. Thousands of lncRNAs are implicated as key players in cellular pathways and cancer biology. However, the structure–function relationships of these novel biomolecules are not well understood. Recent structural studies suggest that lncRNAs contain modular structural domains, which play a crucial role in their function. Here, we hypothesized that such structural domains exist in lncTCF7, a conserved lncRNA implicated in the development and progression of several cancers. To understand the structure–function relationship of lncTCF7, we characterized its secondary structure using chemical probing methods. Our model revealed structural domains and conserved regions in lncTCF7. One of the modular domains identified here coincides with a known protein-interacting domain. The model reported herein is, to our knowledge, the first structural model of lncTCF7 and thus will serve to direct future studies that will provide fundamental insights into the function of this lncRNA.

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In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment

Blood ◽

10.1182/blood.v95.3.731.003k28_731_737 ◽

2000 ◽

Vol 95 (3) ◽

pp. 731-737 ◽

Cited By ~ 58

Author(s):

C. Cobaleda ◽

I. Sánchez-Garcı́a

Keyword(s):

Cancer Treatment ◽

Chromosomal Abnormalities ◽

Human Cancer ◽

Rnase P ◽

Catalytic Rna ◽

Fusion Genes ◽

Novel Approach ◽

Chimeric Molecules

One major obstacle to the effective treatment of cancer is to distinguish between tumor cells and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities are ideal therapeutic targets because they are unique to the disease. We describe the use of a novel approach based on the catalytic RNA subunit of RNase P to destroy specifically the tumor-specific fusion genes created as a result of chromosome abnormalities. Using as a target model the abnormal BCR-ABL p190 and p210 products, we constructed M1-RNA with guide sequences that recognized the oncogenic messengers at the fusion point (M1-p190-GS and M1-p210-GS). To test the effectiveness and the specificity of M1-p190-GS and M1-p210-GS, we studied in vitro and in vivo effects of these RNA enzymes againstBCR-ABLp190 andBCR-ABLp210, bearing in mind that both fusion genes share the ABL sequence but differ in the sequence coming from the BCR gene. We showed that M1-p190-GS and M1-p210-GS can act as sequence-specific endonucleases and can exclusively cleave target RNA that forms a base pair with the guide sequence (GS). We also demonstrated that when M1-p190-GS and M1-p210-GS were expressed in proper mammalian cell models, they abolished the effect of BCR-ABL by specifically decreasing the amount of the target BCR-ABL mRNA and preventing the function of theBCR-ABL oncogenes. These data clearly demonstrate the usefulness of the catalytic activity of M1-GS RNA to cleave specifically the chimeric molecules created by chromosomal abnormalities in human cancer and to represent a novel approach to cancer treatment.

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RNase P inhibitors identified as aggregators

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00300-21 ◽

2021 ◽

Author(s):

Isabell Schencking ◽

Eva M. Schäfer ◽

J. H. William Scanlan ◽

Benjamin M. Wenzel ◽

Rolf E. Emmerich ◽

...

Keyword(s):

High Throughput ◽

Thermotoga Maritima ◽

Rnase P ◽

Inhibition Zone ◽

Model Systems ◽

Catalytic Rna ◽

Plant Origin ◽

Essential Enzyme ◽

P Depletion ◽

Specific Inhibitors

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 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 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 Mg2+ ions. These findings were deduced from solubility analyses by microscopy and 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 non-specific 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 Pan-assay interference compound s (PAINS).

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