Efficient system for upstream mRNA trans-splicing to generate covalent, head-to-tail, protein multimers
| Autor: | Bryant Yu, Satoshi Ishimaru, Mary Lou Beermann, Swetha Duraiswamy, Sachiko Homma, Jeffrey Boone Miller, Hayato Takeda, Hiroaki Mitsuhashi, Frederick M. Boyce, Kevin Liu |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Trans-splicing lcsh:Medicine Article Epitope Trans-Splicing 03 medical and health sciences 0302 clinical medicine Plasmid Complementary DNA Humans Coding region splice RNA Messenger lcsh:Science Homeodomain Proteins Messenger RNA Multidisciplinary Chemistry lcsh:R Cell biology HEK293 Cells 030104 developmental biology lcsh:Q Genetic Engineering 030217 neurology & neurosurgery HeLa Cells Plasmids Binding domain |
| Zdroj: | Scientific Reports, Vol 9, Iss 1, Pp 1-15 (2019) Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | We present a plasmid-based system in which upstream trans-splicing efficiently generates mRNAs that encode head-to-tail protein multimers. In this system, trans-splicing occurs between one of two downstream splice donors in the sequence encoding a C-terminal V5 epitope tag and an upstream splice acceptor in the 5′ region of the pCS2(+) host plasmid. Using deletion and fusion constructs of the DUX4 protein as an example, we found that this system produced trans-spliced mRNAs in which coding regions from independent transcripts were fused in phase such that covalent head-to-tail protein multimers were translated. For a cDNA of ~450 bp, about half of the expressed proteins were multimeric, with the efficiency of trans-splicing and extent of multimer expression decreasing as cDNA length increased. This system generated covalent heterodimeric proteins upon co-transfections of plasmids encoding separate proteins and did not require a long complementary binding domain to position mRNAs for trans-splicing. This plasmid-based trans-splicing system is adaptable to multiple gene delivery systems, and it presents new opportunities for investigating molecular mechanisms of trans-splicing, generating covalent protein multimers with novel functions within cells, and producing mRNAs encoding large proteins from split precursors. |
| Databáze: | OpenAIRE |
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