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) |