hnRNP G/RBMX enhances HPV16 E2 mRNA splicing through a novel splicing enhancer and inhibits production of spliced E7 oncogene mRNAs

Human papillomavirus type 16 (HPV16) E2 is an essential HPV16 protein. We have investigated how HPV16 E2 expression is regulated and have identifed a splicing enhancer that is required for production of HPV16 E2 mRNAs. This uridine-less splicing enhancer sequence (ACGAGGACGAGGACAAGGA) contains 84% adenosine and guanosine and 16% cytosine and consists of three 'AC(A/G)AGG'-repeats. Mutational inactivation of the splicing enhancer reduced splicing to E2-mRNA specific splice site SA2709 and resulted in increased levels of unspliced E1-encoding mRNAs. The splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G that promoted splicing to SA2709 and enhanced E2 mRNA production. The splicing-enhancing function of hnRNP G mapped to amino acids 236-286 of hnRNP G that were also shown to interact with splicing factor U2AF65. The interactions between hnRNP G and HPV16 E2 mRNAs and U2AF65 increased in response to keratinocyte differentiation as well as by the induction of the DNA damage response (DDR). The DDR reduced sumoylation of hnRNP G and pharmacological inhibition of sumoylation enhanced HPV16 E2 mRNA splicing and interactions between hnRNP G and E2 mRNAs and U2AF65. Intriguingly, hnRNP G also promoted intron retention of the HPV16 E6 coding region thereby inhibiting production of spliced E7 oncogene mRNAs.