Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo

Abstract

The 5 0 untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment and c-Myc translation can be initiated by cap-independent as well as cap-dependent mechanisms. In contrast to the process of cap-dependent initiation, the trans-acting factor requirements for cellular internal ribosome entry are poorly understood. Here, we show that members of the poly (rC) binding protein family, poly (rC) binding protein 1 (PCBP1), poly (rC) binding protein 2 (PCBP2) and hnRNPK were able to activate the IRES in vitro up to threefold when added in combination with upstream of N-ras and unr-interacting protein. The interactions of PCBP1, PCBP2 and hnRNPK with c-myc-IRES-RNA were shown to be specific by ultraviolet crosslinking analysis and electrophoretic mobility shift assays, while immunoprecipitation of the three proteins using specific antibodies followed by reverse transcriptase-polymerase chain reaction showed that they were able to bind c-myc mRNA. c-myc-IRES-mediated translation from the reporter vector was stimulated by cotransfection of plasmids encoding PCBP1, PCBP2 and hnRNPK. Interestingly, the mutated version of the c-myc IRES that is prevalent in patients with multiple myeloma bound hnRNPK more efficiently in vitro and was stimulated by hnRNPK to a greater extent in vivo.

Thymidine phosphorylase mRNA stability and protein levels are increased through ERK-mediated cytoplasmic accumulation of hnRNP K in nasopharyngeal carcinoma cells

“…Cytoplasmic localization of hnRNP K is required for BCR/ABL oncogenic activity in chronic myelogenous leukemia (Notari et al, 2006), and has been shown to promote cellular migration in fibrosarcoma cells (Inoue et al, 2007). Cytoplasmic hnRNP K also regulates posttranscriptional activity; it has been shown to directly stimulate IRES-dependent c-myc translation (Evans et al, 2003;Notari et al, 2006) and stabilize the gastrin mRNA . Accordingly, cytoplasmic hnRNP K may elevate tumorigenic potential by altering the expression of its target genes at the posttranscriptional level.…”

“…The tumorigenic activity of hnRNP K is conferred through its ability to increase proliferation (Lynch et al, 2005), clonogenic potential (Notari et al, 2006) and metastasis (Inoue et al, 2007). These effects may be due, at least in part, to the ability of hnRNP K to upregulate c-myc expression through the c-myc internal ribosome entry segment (IRES; Evans et al, 2003;Notari et al, 2006). hnRNP K is a nucleocytoplasmic shuttling protein and primarily located in the nucleus (Michael et al, 1997); however, cytoplasmic accumulation of hnRNP K via ERKmediated phosphorylation of hnRNP K serine-284 and -353 has been reported in cervical carcinoma HeLa cells (Habelhah et al, 2001) and chronic myelogenous leukemia cells (Notari et al, 2006).…”

AKT activity regulates the ability of mTOR inhibitors to prevent angiogenesis and VEGF expression in multiple myeloma cells

“…These factors bind to RNA at specific sites in the 5 0 -UTR and induce conformational changes that facilitate recruitment of the ribosome to the IRES (Stoneley et al, 2000;Evans et al, 2003). Thus, one possible explanation for AKT's regulatory role is that it affects expression or binding of specific ITAFs to the VEGF IRES.…”

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

“…In combination with PCBP1, these interact specifically with regions of the c-myc IRES, and increase internal translation initiation. 61,62 Since c-myc translation also occurs via cap-dependent initiation, the 5 0 UTR needs to be sufficiently flexible to allow ribosome scanning to the initiation AUG. The ITAFs in this case may be required to hold the RNA in the correct conformation for internal recruitment of the ribosome.…”