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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2022-12-01
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| Series: | RNA Biology |
| Subjects: | |
| Online Access: | http://dx.doi.org/10.1080/15476286.2022.2027659 |
| _version_ | 1797403951437447168 |
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| author | Merel Derksen Vicky Mertens Eline A. Visser Janine Arts Wilma Vree Egberts Ger J. M. Pruijn |
| author_facet | Merel Derksen Vicky Mertens Eline A. Visser Janine Arts Wilma Vree Egberts Ger J. M. Pruijn |
| author_sort | Merel Derksen |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-09T02:47:01Z |
| format | Article |
| id | doaj.art-1ef8102a5f35469ebbed264ceedfbd6a |
| institution | Directory Open Access Journal |
| issn | 1547-6286 1555-8584 |
| language | English |
| last_indexed | 2024-03-09T02:47:01Z |
| publishDate | 2022-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | RNA Biology |
| spelling | doaj.art-1ef8102a5f35469ebbed264ceedfbd6a2023-12-05T16:09:51ZengTaylor & Francis GroupRNA Biology1547-62861555-85842022-12-0119130531210.1080/15476286.2022.20276592027659A novel experimental approach for the selective isolation and characterization of human RNase MRPMerel Derksen0Vicky Mertens1Eline A. Visser2Janine Arts3Wilma Vree Egberts4Ger J. M. Pruijn5Radboud UniversityRadboud UniversityRadboud UniversityRadboud UniversityRadboud UniversityRadboud UniversityRNase 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.http://dx.doi.org/10.1080/15476286.2022.2027659rnase mrprnase pribonucleoproteinrna aptamer |
| spellingShingle | Merel Derksen Vicky Mertens Eline A. Visser Janine Arts Wilma Vree Egberts Ger J. M. Pruijn A novel experimental approach for the selective isolation and characterization of human RNase MRP RNA Biology rnase mrp rnase p ribonucleoprotein rna aptamer |
| 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 | rnase mrp rnase p ribonucleoprotein rna aptamer |
| url | http://dx.doi.org/10.1080/15476286.2022.2027659 |
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