Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A.
RNA helicase A (RHA), a member of the DEXH box helicase family of proteins, is an integral component of protein complexes that regulate transcription and splicing. The EWS-FLI1 oncoprotein is expressed as a result of the chromosomal translocation t(11;22) that occurs in patients with the Ewing's sarcoma family of tumors (ESFT). Using phage display library screening, we identified an EWS-FLI1 binding peptide containing homology to RHA. ESFT cell lines and patient tumors highly expressed RHA. GST pull-down and ELISA assays showed that EWS-FLI1 specifically bound RHA fragment amino acids 630 to 1020, which contains the peptide region discovered by phage display. Endogenous RHA was identified in a protein complex with EWS-FLI1 in ESFT cell lines. Chromatin immunoprecipitation experiments showed both EWS-FLI1 and RHA bound to EWS-FLI1 target gene promoters. RHA stimulated the transcriptional activity of EWS-FLI1 regulated promoters, including Id2, in ESFT cells. In addition, RHA expression in mouse embryonic fibroblast cells stably transfected with EWS-FLI1 enhanced the anchorage-independent phenotype above that with EWS-FLI1 alone. These results suggest that RHA interacts with EWS-FLI1 as a transcriptional cofactor to enhance its function.
Toretsky
Jeffrey A
JA
Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA. jat42@georgetown.edu
Erkizan
Verda
V
Levenson
Amy
A
Abaan
Ogan D
OD
Parvin
Jeffrey D
JD
Cripe
Timothy P
TP
Rice
Anna M
AM
Lee
Sean Bong
SB
Uren
Aykut
A
D000818:Animals; D001324:Autoantigens / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D048708:Cell Growth Processes / Q000502:physiology; D045744:Cell Line, Tumor; D053487:DEAD-box RNA Helicases; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics / Q000378:metabolism*; D020365:RNA Helicases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D034802:RNA-Binding Protein EWS; D011994:Recombinant Proteins / Q000378:metabolism; D012512:Sarcoma, Ewing / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014183:Transplantation, Heterologous
D001324:Autoantigens; C106031:EWS-FLI fusion protein; D009363:Neoplasm Proteins; D015514:Oncogene Proteins, Fusion; D019151:Peptide Library; D010455:Peptides; D051789:Proto-Oncogene Protein c-fli-1; D034802:RNA-Binding Protein EWS; D011994:Recombinant Proteins; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases