Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo.
The 5' 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.
Evans
Joanne R
JR
Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK.
Mitchell
Sally A
SA
Spriggs
Keith A
KA
Ostrowski
Jerzy
J
Bomsztyk
Karol
K
Ostarek
Dirk
D
Willis
Anne E
AE
D001665:Binding Sites; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000235:genetics / Q000378:metabolism*; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008967:Molecular Biology / Q000379:methods; D005810:Multigene Family; D009101:Multiple Myeloma / Q000235:genetics; D009154:Mutation; D014176:Protein Biosynthesis; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins; D012045:Regulatory Sequences, Nucleic Acid; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C000715470:MAS1 protein, human; C094604:PCBP1 protein, human; C501903:PCBP2 protein, human; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; D016601:RNA-Binding Proteins; D014157:Transcription Factors