Spurious splicing within the XIAP 5′ UTR occurs in the Rluc/Fluc but not the βgal/CAT bicistronic reporter system

Holčı́k, Martin; Graber, Tyson E.; Lewis, Stephen M.; Lefebvre, Charles; LaCasse, Eric C.; Baird, Stephen

doi:10.1261/rna.2158605

Cited by 62 publications

(64 citation statements)

References 16 publications

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“…DNA transfections were performed as previously reported (7). The reporter gene constructs containing the p27 Kip1 , XIAP, or Bcl-xl 5′ UTR were previously described (38,80,81). Quantification of firefly and renilla luciferase activity was performed with the Dual-Luciferase Reporter Assay System (Promega).…”

Section: Methodsmentioning

confidence: 99%

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Stat1 stimulates cap-independent mRNA translation to inhibit cell proliferation and promote survival in response to antitumor drugs

Wang

Patsis

Koromilas

2015

Proc. Natl. Acad. Sci. U.S.A.

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The signal transducer and activator of transcription 1 (Stat1) functions as a tumor suppressor via immune regulatory and cell-autonomous pathways. Herein, we report a previously unidentified cell-autonomous Stat1 function, which is its ability to exhibit both antiproliferative and prosurvival properties by facilitating translation of mRNAs encoding for the cyclin-dependent kinase inhibitor p27Kip1 and antiapoptotic proteins X-linked inhibitor of apoptosis and B-cell lymphoma xl. Translation of the select mRNAs requires the transcriptional function of Stat1, resulting in the up-regulation of the p110γ subunit of phosphoinositide 3-kinase (PI3K) class IB and increased expression of the translational repressor translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). Increased PI3Kγ signaling promotes the degradation of the eIF4A inhibitor programmed cell death protein 4, which favors the cap-independent translation of the select mRNAs under conditions of general inhibition of protein synthesis by up-regulated eIF4E-binding protein 1. As such, Stat1 inhibits cell proliferation but also renders cells increasingly resistant to antiproliferative effects of pharmacological inhibitors of PI3K and/or mammalian target of rapamycin. Stat1 also protects Ras-transformed cells from the genotoxic effects of doxorubicin in culture and immune-deficient mice. Our findings demonstrate an important role of mRNA translation in the cell-autonomous Stat1 functions, with implications in tumor growth and treatment with chemotherapeutic drugs.

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Section: Methodsmentioning

confidence: 99%

“…Quantification of firefly and renilla luciferase activity was performed with the Dual-Luciferase Reporter Assay System (Promega). Quantification of β-galactosidase and chloramphenicol acetyltransferase (CAT) activity was performed as described (81).…”

Section: Methodsmentioning

confidence: 99%

Stat1 stimulates cap-independent mRNA translation to inhibit cell proliferation and promote survival in response to antitumor drugs

Wang

Patsis

Koromilas

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…35,36 To exclude the possibility of artificial splicing, we then examined the RNA products of the pβgal/AMAP1/CAT plasmid, using a CAT DNA fragment as a probe. We detected a single we next sought to map the sequence responsible for the IRES activity by generating deletion mutants of this 5'-UTR (Fig.…”

Section: Resultsmentioning

confidence: 99%

IRES-mediated translational control of AMAP1 expression during differentiation of monocyte U937 cells

Miyata¹,

Raven²,

Baltzis³

et al. 2008

Cell Cycle

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Global control of mRNA translation plays key roles in cell regulation, including growth, differentiation and apoptosis. Human monocyte-like U937 cells differentiate into macrophage-like cells upon 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, a process which is known to be accompanied with a large decrease in general protein synthesis. Here, we found that protein levels of AMAP1 (also called ASAP1 or DDEF1), a GTPase-activating protein for Arf GTPases, increase several fold during U937 cell differentiation. This increase was not accompanied with a notable increase in the AMAP1 gene transcript, nor seemed to be due to 5'-Cap-dependent mRNA translational activities in differentiated U937 cells. We identified the 5'-untranslated region (5'-UTR) of AMAP1 mRNA, and found that this 5'-UTR exhibits significant internal ribosome entry site (IRES)-dependent translational activity in differentiated U937 cells, but not in undifferentiated cells. Our results indicate that monocyte differentiation involves enhancement of IRES activity, by which protein levels of AMAP1 are primarily upregulated.

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“…To this end, we performed transient transfection assays of di-cistronic constructs bearing the 5 0 UTR of either BCL-XL or XIAP mRNA between the b-galactosidase and CAT reporter genes. These dicistronic constructs were previously demonstrated to be devoid of splice-acceptor sites involved in spurious IRES activity (21). The di-cistronic reporter assays revealed that IRES activity of each mRNA was more highly increased in STAT1-proficient than deficient cells (Fig.…”

Section: Stat1 Promotes Pi3k Signaling and Expression Of Xiap And Bclmentioning

confidence: 91%

“…Quantification of b-galactosidase and chloramphenicol acetyltransferase (CAT) activity was performed as described previously (21).…”

Section: Transfections and Luciferase Reporter Gene Assaysmentioning

confidence: 99%

STAT1 Promotes KRAS Colon Tumor Growth and Susceptibility to Pharmacological Inhibition of Translation Initiation Factor eIF4A

Wang

Darini

Désaubry

et al. 2016

Molecular Cancer Therapeutics

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The transcription factor STAT1 displays antitumor functions for certain forms of cancer via immunoregulatory and cellautonomous pathways. Paradoxically, STAT1 can promote the survival of different tumor types treated with chemotherapeutic drugs through mechanisms that are not clearly defined. Herein, we demonstrate that STAT1 displays prosurvival effects in human KRAS colon tumor cells by regulating pathways that converge on the initiation of mRNA translation. Specifically, STAT1 increases PI3K class IB signaling and promotes the downregulation of the programmed cell death protein 4 (PDCD4), a protein with tumor-suppressive properties. PDCD4 downregulation by STAT1 increases the activity of the translation initiation factor eIF4A, which facilitates the capindependent translation of mRNAs encoding for the antiapoptotic XIAP and BCL-XL in colon tumors with mutated but not normal KRAS. Genetic inactivation of STAT1 impairs the tumorigenic potency of human KRAS colon tumor cells and renders them resistant to the antitumor effects of the pharmacologic inhibition of eIF4A in culture and immunodeficient mice. Our data demonstrate an important connection between mRNA translation and KRAS tumorigenesis under the control of STAT1, which can determine the susceptibility of KRAS tumors to pharmacologic inhibition of mRNA translation initiation. Mol Cancer Ther; 15(12); 3055-63. Ó2016 AACR.

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Spurious splicing within the XIAP 5′ UTR occurs in the Rluc/Fluc but not the βgal/CAT bicistronic reporter system

Cited by 62 publications

References 16 publications

Stat1 stimulates cap-independent mRNA translation to inhibit cell proliferation and promote survival in response to antitumor drugs

Stat1 stimulates cap-independent mRNA translation to inhibit cell proliferation and promote survival in response to antitumor drugs

IRES-mediated translational control of AMAP1 expression during differentiation of monocyte U937 cells

STAT1 Promotes KRAS Colon Tumor Growth and Susceptibility to Pharmacological Inhibition of Translation Initiation Factor eIF4A

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