RRC ID 54880
著者 Mitsuhashi H, Homma S, Beermann ML, Ishimaru S, Takeda H, Yu BK, Liu K, Duraiswamy S, Boyce FM, Miller JB.
タイトル Efficient system for upstream mRNA trans-splicing to generate covalent, head-to-tail, protein multimers.
ジャーナル Sci Rep
Abstract 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.
巻・号 9(1)
ページ 2274
公開日 2019-2-19
DOI 10.1038/s41598-018-36684-7
PII 10.1038/s41598-018-36684-7
PMID 30783185
PMC PMC6381186
MeSH Genetic Engineering* HEK293 Cells HeLa Cells Homeodomain Proteins / genetics Homeodomain Proteins / metabolism Humans Plasmids / genetics* Plasmids / metabolism RNA, Messenger* / biosynthesis RNA, Messenger* / genetics Trans-Splicing*
IF 4.011
引用数 0
リソース情報
ヒト・動物細胞 HeLa(RCB0007)