A novel experimental approach for the selective isolation and characterization of human RNase MRP

RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of...

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Autores principales: Derksen, Merel, Mertens, Vicky, Visser, Eline A., Arts, Janine, Vree Egberts, Wilma, Pruijn, Ger J. M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8820802/
https://www.ncbi.nlm.nih.gov/pubmed/35129080
http://dx.doi.org/10.1080/15476286.2022.2027659
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author Derksen, Merel
Mertens, Vicky
Visser, Eline A.
Arts, Janine
Vree Egberts, Wilma
Pruijn, Ger J. M.
author_facet Derksen, Merel
Mertens, Vicky
Visser, Eline A.
Arts, Janine
Vree Egberts, Wilma
Pruijn, Ger J. M.
author_sort Derksen, Merel
collection PubMed
description RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of RNase MRP is desired to be able to study its biochemical function, composition and activity in both healthy and disease situations. Due to the high similarity with RNase P, a method to specifically isolate the RNase MRP complex is currently lacking. By fusing a streptavidin-binding RNA aptamer, the S1m-aptamer, to the RNase MRP RNA we have been able to compare the relative expression levels of wildtype and mutant MRP RNAs. Moreover, we were able to isolate active RNase MRP complexes. We observed that mutant MRP RNAs are expressed at lower levels and have lower catalytic activity compared to the wildtype RNA. The observation that a single nucleotide substitution at position 40 in the P3 domain but not in other domains of RNase MRP RNA severely reduced the binding of the Rpp25 protein subunit confirmed that the P3 region harbours the main binding site for this protein. Altogether, this study shows that the RNA aptamer tagging approach can be used to identify RNase MRP substrates, but also to study the effect of mutations on MRP RNA expression levels and RNase MRP composition and endoribonuclease activity.
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spelling pubmed-88208022022-02-08 A novel experimental approach for the selective isolation and characterization of human RNase MRP Derksen, Merel Mertens, Vicky Visser, Eline A. Arts, Janine Vree Egberts, Wilma Pruijn, Ger J. M. RNA Biol Research Paper RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of RNase MRP is desired to be able to study its biochemical function, composition and activity in both healthy and disease situations. Due to the high similarity with RNase P, a method to specifically isolate the RNase MRP complex is currently lacking. By fusing a streptavidin-binding RNA aptamer, the S1m-aptamer, to the RNase MRP RNA we have been able to compare the relative expression levels of wildtype and mutant MRP RNAs. Moreover, we were able to isolate active RNase MRP complexes. We observed that mutant MRP RNAs are expressed at lower levels and have lower catalytic activity compared to the wildtype RNA. The observation that a single nucleotide substitution at position 40 in the P3 domain but not in other domains of RNase MRP RNA severely reduced the binding of the Rpp25 protein subunit confirmed that the P3 region harbours the main binding site for this protein. Altogether, this study shows that the RNA aptamer tagging approach can be used to identify RNase MRP substrates, but also to study the effect of mutations on MRP RNA expression levels and RNase MRP composition and endoribonuclease activity. Taylor & Francis 2022-02-06 /pmc/articles/PMC8820802/ /pubmed/35129080 http://dx.doi.org/10.1080/15476286.2022.2027659 Text en © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Paper
Derksen, Merel
Mertens, Vicky
Visser, Eline A.
Arts, Janine
Vree Egberts, Wilma
Pruijn, Ger J. M.
A novel experimental approach for the selective isolation and characterization of human RNase MRP
title A novel experimental approach for the selective isolation and characterization of human RNase MRP
title_full A novel experimental approach for the selective isolation and characterization of human RNase MRP
title_fullStr A novel experimental approach for the selective isolation and characterization of human RNase MRP
title_full_unstemmed A novel experimental approach for the selective isolation and characterization of human RNase MRP
title_short A novel experimental approach for the selective isolation and characterization of human RNase MRP
title_sort novel experimental approach for the selective isolation and characterization of human rnase mrp
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8820802/
https://www.ncbi.nlm.nih.gov/pubmed/35129080
http://dx.doi.org/10.1080/15476286.2022.2027659
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