Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform
Autoři:
Natalia Berges aff001; Meh Sameen Nawaz aff001; Tuva Børresdatter Dahl aff001; Lars Hagen aff003; Magnar Bjørås aff001; Jon K. Laerdahl aff001; Ingrun Alseth aff001
Působiště autorů:
Department of Microbiology, Oslo University Hospital Rikshospitalet and University of Oslo, Oslo, Norway
aff001; Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet and Institute of Clinical Medicine, University of Oslo, Oslo, Norway
aff002; Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway
aff003; PROMEC Core Facility for Proteomics and Modomics, Norwegian University of Science and Technology and Central Norway Regional Health Authority, Trondheim, Norway
aff004
Vyšlo v časopise:
PLoS ONE 14(11)
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pone.0225081
Souhrn
Endonuclease V (ENDOV) is a ribonuclease with affinity for inosine which is the deamination product of adenosine. The genomes of most organisms, including human, encode ENDOV homologs, yet knowledge about in vivo functions and gene regulation is sparse. To contribute in this field, we analyzed mRNA and protein expression of human ENDOV (hENDOV). Analyses of public sequence databases revealed numerous hENDOV transcript variants suggesting extensive alternative splicing. Many of the transcripts lacked one or more exons corresponding to conserved regions of the ENDOV core domain, suggesting that these transcripts do not encode for active proteins. Three complete transcripts were found with open reading frames encoding 282, 308 and 309 amino acids, respectively. Recombinant hENDOV 308 and hENDOV 309 share the same cleavage activity as hENDOV 282 which is the variant that has been used in previous studies of hENDOV. However, hENDOV 309 binds inosine-containing RNA with stronger affinity than the other isoforms. Overexpressed GFP-fused isoforms were found in cytoplasm, nucleoli and arsenite induced stress granules in human cells as previously reported for hENDOV 282. RT-qPCR analysis of the 3’-termini showed that hENDOV 308 and hENDOV 309 transcripts are more abundant than hENDOV 282 transcripts in immortalized cell lines, but not in primary cells, suggesting that cells regulate hENDOV mRNA expression. In spite of the presence of all three full-length transcripts, mass spectrometry analyses identified peptides corresponding to the hENDOV 309 isoform only. This result suggests that further studies of human ENDOV should rather encompass the hENDOV 309 isoform.
Klíčová slova:
Messenger RNA – Polymerase chain reaction – 293T cells – Macrophages – Immunoprecipitation – Recombinant proteins – Inosine – Ribozymes
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