In vitro studies of disease-linked variants of human tRNA nucleotidyltransferase reveal decreased thermal stability and altered catalytic activity |
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| Affiliation: | 1. Carmen and Ann Adams Department of Pediatrics, Children''s Hospital of Michigan, Wayne State University School of Medicine, Detroit, MI 48201, USA;2. Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI 48201, USA;3. Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, USA;4. Molecular Genetics Laboratory, Detroit Medical Center University Laboratories, Detroit, MI 48201, USA;5. The Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201, USA;6. Genetics Research Laboratory of the Department of Research Administration, Henry Ford Hospital, Detroit, MI 48202, USA;1. Genomic Diagnostics Laboratory, Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, St Mary''s Hospital, Oxford Road, Manchester M13 9WL, UK;2. Manchester Adult Cystic Fibrosis Centre, University Hospital of South Manchester, Southmoor Road, Manchester, M23 9LT, UK;3. Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, Manchester Academic Health Science Centre, University of Manchester, Manchester M23 9LT, UK |
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| Abstract: | Mutations in the human TRNT1 gene encoding tRNA nucleotidyltransferase (tRNA-NT), an essential enzyme responsible for addition of the CCA (cytidine-cytidine-adenosine) sequence to the 3′-termini of tRNAs, have been linked to disease phenotypes including congenital sideroblastic anemia with B-cell immunodeficiency, periodic fevers and developmental delay (SIFD) or retinitis pigmentosa with erythrocyte microcytosis. The effects of these disease-linked mutations on the structure and function of tRNA-NT have not been explored. Here we use biochemical and biophysical approaches to study how five SIFD-linked amino acid substitutions (T154I, M158V, L166S, R190I and I223T), residing in the N-terminal head and neck domains of the enzyme, affect the structure and activity of human tRNA-NT in vitro. Our data suggest that the SIFD phenotype is linked to poor stability of the T154I and L166S variant proteins, and to a combination of reduced stability and altered catalytic efficiency in the M158 V, R190I and I223T variants. |
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