Two complementing in vivo selection systems based on CCA-trimming exonucleases as a tool to monitor, select and evaluate enzymatic features of tRNA nucleotidyltransferases
tRNA nucleotidyltransferase represents a ubiquitous and essential activity that adds the indispensable CCA triplet to the 3’-end of tRNAs. To fulfill this function, the enzyme contains a set of highly conserved motifs whose coordinated interplay is crucial for the sequence-specific CCA polymerizatio...
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| Language: | English |
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Taylor & Francis Group
2025-12-01
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| Series: | RNA Biology |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/15476286.2025.2453963 |
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| author | Karolin Wellner Josefine Gnauck Dorian Bernier Stephan H. Bernhart Heike Betat Mario Mörl |
| author_facet | Karolin Wellner Josefine Gnauck Dorian Bernier Stephan H. Bernhart Heike Betat Mario Mörl |
| author_sort | Karolin Wellner |
| collection | DOAJ |
| description | tRNA nucleotidyltransferase represents a ubiquitous and essential activity that adds the indispensable CCA triplet to the 3’-end of tRNAs. To fulfill this function, the enzyme contains a set of highly conserved motifs whose coordinated interplay is crucial for the sequence-specific CCA polymerization. In the human enzyme, alterations within these regions have been shown to lead to the manifestation of disease. Recently, we developed an in vivo screening system that allows for the selection and analysis of tRNA nucleotidyltransferase variants by challenging terminal AMP incorporation into tRNA during induced RNase T-catalyzed CCA-decay. Here, we extend this method for screening of full CCA-end repair by utilizing the CCA-trimming activity of exonuclease LCCR4. To demonstrate the combined potential of these two in vivo selection systems, we applied a semi-rational library design to investigate the mode of operation of catalytically important motifs in the human CCA-adding enzyme. This approach revealed unexpected requirements for amino acid composition in two motifs and gives new insights into the mechanism of CCA addition. The data show the potential of these RNase-based screening systems, as they allow the detection of enzyme variations that would not have been identified by a conventional rational approach. Furthermore, the combination of both RNase T and LCCR4 systems can be used to investigate and dissect the effects of pathogenic mutations on C- and A-addition. |
| format | Article |
| id | doaj-art-6474c735b7a34ef0a1ea34f91796d399 |
| institution | Kabale University |
| issn | 1547-6286 1555-8584 |
| language | English |
| publishDate | 2025-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | RNA Biology |
| spelling | doaj-art-6474c735b7a34ef0a1ea34f91796d3992025-01-30T06:03:12ZengTaylor & Francis GroupRNA Biology1547-62861555-85842025-12-0122111410.1080/15476286.2025.2453963Two complementing in vivo selection systems based on CCA-trimming exonucleases as a tool to monitor, select and evaluate enzymatic features of tRNA nucleotidyltransferasesKarolin Wellner0Josefine Gnauck1Dorian Bernier2Stephan H. Bernhart3Heike Betat4Mario Mörl5Institute for Biochemistry, Leipzig University, Leipzig, GermanyInstitute for Biochemistry, Leipzig University, Leipzig, GermanyInstitute for Biochemistry, Leipzig University, Leipzig, GermanyBioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, Leipzig University, Leipzig, GermanyInstitute for Biochemistry, Leipzig University, Leipzig, GermanyInstitute for Biochemistry, Leipzig University, Leipzig, GermanytRNA nucleotidyltransferase represents a ubiquitous and essential activity that adds the indispensable CCA triplet to the 3’-end of tRNAs. To fulfill this function, the enzyme contains a set of highly conserved motifs whose coordinated interplay is crucial for the sequence-specific CCA polymerization. In the human enzyme, alterations within these regions have been shown to lead to the manifestation of disease. Recently, we developed an in vivo screening system that allows for the selection and analysis of tRNA nucleotidyltransferase variants by challenging terminal AMP incorporation into tRNA during induced RNase T-catalyzed CCA-decay. Here, we extend this method for screening of full CCA-end repair by utilizing the CCA-trimming activity of exonuclease LCCR4. To demonstrate the combined potential of these two in vivo selection systems, we applied a semi-rational library design to investigate the mode of operation of catalytically important motifs in the human CCA-adding enzyme. This approach revealed unexpected requirements for amino acid composition in two motifs and gives new insights into the mechanism of CCA addition. The data show the potential of these RNase-based screening systems, as they allow the detection of enzyme variations that would not have been identified by a conventional rational approach. Furthermore, the combination of both RNase T and LCCR4 systems can be used to investigate and dissect the effects of pathogenic mutations on C- and A-addition.https://www.tandfonline.com/doi/10.1080/15476286.2025.2453963CCA-adding enzymetRNA nucleotidyltransferaseCCA-trimming exonucleaseRNase LCCR4RNase Tin vivo screening of CCA-adding activities |
| spellingShingle | Karolin Wellner Josefine Gnauck Dorian Bernier Stephan H. Bernhart Heike Betat Mario Mörl Two complementing in vivo selection systems based on CCA-trimming exonucleases as a tool to monitor, select and evaluate enzymatic features of tRNA nucleotidyltransferases RNA Biology CCA-adding enzyme tRNA nucleotidyltransferase CCA-trimming exonuclease RNase LCCR4 RNase T in vivo screening of CCA-adding activities |
| title | Two complementing in vivo selection systems based on CCA-trimming exonucleases as a tool to monitor, select and evaluate enzymatic features of tRNA nucleotidyltransferases |
| title_full | Two complementing in vivo selection systems based on CCA-trimming exonucleases as a tool to monitor, select and evaluate enzymatic features of tRNA nucleotidyltransferases |
| title_fullStr | Two complementing in vivo selection systems based on CCA-trimming exonucleases as a tool to monitor, select and evaluate enzymatic features of tRNA nucleotidyltransferases |
| title_full_unstemmed | Two complementing in vivo selection systems based on CCA-trimming exonucleases as a tool to monitor, select and evaluate enzymatic features of tRNA nucleotidyltransferases |
| title_short | Two complementing in vivo selection systems based on CCA-trimming exonucleases as a tool to monitor, select and evaluate enzymatic features of tRNA nucleotidyltransferases |
| title_sort | two complementing in vivo selection systems based on cca trimming exonucleases as a tool to monitor select and evaluate enzymatic features of trna nucleotidyltransferases |
| topic | CCA-adding enzyme tRNA nucleotidyltransferase CCA-trimming exonuclease RNase LCCR4 RNase T in vivo screening of CCA-adding activities |
| url | https://www.tandfonline.com/doi/10.1080/15476286.2025.2453963 |
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