Variable requirement for splicing signals for nucleocytoplasmic export of mRNAs

Fu, Litao; Suen, Christine K. M.; Ahmed, Waseem; White, Kenneth

doi:10.1080/15216549700202731

Cited by 2 publications

(2 citation statements)

References 12 publications

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“…GFP-KH4-TCP80/NF90 (where NF90 stands for nuclear factor 90) was generated from a GFP-KH4 construct by insertion of full-length TCP80/NF90 downstream of KH4. Human H19 (H19C) was inserted into an intronless version of pcDNA1 [15]. In H19C-IGF-IIsegC (where IGF stands for insulin-like growth factor) and H19C-A11, the high-affinity IMP1-binding sites from IGF-II leader 3 segment C [4] and A11 were cloned into pcDNA1-H19C respectively.…”

Section: Generation Of Dna Constructs and Recombinant Proteinsmentioning

confidence: 99%

Nuclear transit of human zipcode-binding protein IMP1

Nielsen

Adolph

Meyts

et al. 2003

Biochemical Journal

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The human IMPs (insulin-like growth factor II mRNA-binding proteins) belong to a vertebrate zipcode-binding protein family consisting of two RNA recognition motifs and four K homology domains and have been implicated in cytoplasmic mRNA localization, turnover and translational control. In the present study, we show that IMP1 is capable of translocating into nuclei of NIH 3T3 fibroblasts and its immunoreactivity is present in the nuclei of human spermatogenic cells. IMP1 does not contain a simple import signal, but nuclear entry was facilitated by disruption of RNA binding and cytoplasmic granule formation. IMP1 contains two NESs (nuclear export signals) within the RNA-binding K homology domains 2 and 4. The former is a leucine-rich leptomycin B-sensitive NES, whereas the latter is a leptomycin B-insensitive NES. Taken together, these results indicate that IMP1 may attach to its target mRNAs in the nucleus and thereby define the cytoplasmic fate of the transcripts.

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Section: Generation Of Dna Constructs and Recombinant Proteinsmentioning

confidence: 99%

Nuclear transit of human zipcode-binding protein IMP1

Nielsen

Adolph

Meyts

et al. 2003

Biochemical Journal

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“…The expression of a GOI is enhanced when introns are transcribed with the exons of a protein-coding RNA; because nascent RNAs that undergo splicing are more effectively coupled to the mRNA export machinery than are nascent RNAs that do not contain introns [47-49]. Indeed, the EF1A and polyubiquitin promoters are sixfold more active if the first intron within the 5' UTR is present [50,51].…”

Section: Resultsmentioning

confidence: 99%

Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid

Krause¹,

Izotova²,

Ren³

et al. 2011

Stem Cell Res Ther

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IntroductionLocal synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably.MethodsTo follow both the recruitment to tumors and the synthesis of interferon by MSCs, we designed a bicistronic vector system that permits fluorescent visualization of vector-transfected and interferon-producing MSCs. We used Mu-IFNαA cDNA as the first cistron and the cherry fluorescent protein cDNA as the second cistron, whose translation requires the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus 5' untranslated region. Observing inconsistent expression of these cistrons in various vectors and cell lines, especially compared with a control plasmid pmaxGFP, we optimized the expression of this bicistronic message by mutating pcDNA3 to facilitate exchange of the promoter and polyadenylation segments controlling both the gene of interest and the eukaryotic antibiotic resistance gene as well as the eukaryotic antibiotic resistance gene itself, and effectively compare the effects of these exchanges, creating plasmid pc3.5.ResultsMurine MSCs stably and ectopically expressing Mu-IFNαA inhibited the establishment of tumors in homogeneic C57/BL6 mice. Mu-IFNαA expressed from the bicistronic message is fully biologically active, but is expressed at only two-thirds of the level observed from a monocistronic message. Cap-dependent translation is threefold more efficient than IRES-driven translation in 293T, B16, and MSC cell lines. Both efficient expression and good transfection efficiency require strong expression of the gene of interest and a chimeric intron. High doses of Mu-IFNαA within tumors inhibited tumor establishment but may not inhibit tumor growth.ConclusionsOur modified vector and its derived plasmids will find use in stem cell therapeutics, gene expression, mRNA regulation, and transcription regulation. Local release of Mu-IFNαA within tumors may differently affect tumor establishment and tumor growth.

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Variable requirement for splicing signals for nucleocytoplasmic export of mRNAs

Cited by 2 publications

References 12 publications

Nuclear transit of human zipcode-binding protein IMP1

Nuclear transit of human zipcode-binding protein IMP1

Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid

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