Alteration of cyclin D1 transcript elongation by a mutated transcription factor up-regulates the oncogenic D1b splice isoform in cancer

Abstract

Pre-mRNA splicing and polyadenylation are tightly connected to transcription, and transcriptional stimuli and elongation dynamics can affect mRNA maturation. However, whether this regulatory mechanism has a physio/pathological impact is not known. In cancer, where splice variant expression is often deregulated, many mutated oncogenes are transcriptional regulators. In particular, the Ewing sarcoma (EwSa) oncogene, resulting from a fusion of the EWS and FLI1 genes, encodes a well characterized transcription factor. EWS-FLI1 directly stimulates transcription of the CCND1 protooncogene encoding cyclin D1a and a less abundant but more oncogenic splice isoform, D1b. We show that, although both EWS and EWS-FLI1 enhance cyclin D1 gene expression, they regulate the D1b/D1a transcript ratio in an opposite manner. Detailed analyses of RNA polymerase dynamics along the gene and of the effects of an inhibitor of elongation show that EWS-FLI1 favors D1b isoform expression by decreasing the elongation rate, whereas EWS has opposite effects. As a result, the D1b/D1a ratio is elevated in EwSa cell lines and tumors. The endogenous D1b protein is enriched in nuclei, where the oncogenic activity of cyclin D1 is known to occur, and depleting D1b in addition to D1a results in a stronger reduction of EwSa cell growth than depleting D1a only. These data show that elevated expression of a splice isoform in cancer can be due to an alteration of the transcription process by a mutated transcriptional regulator and provide evidence for a physio/pathological impact of the coupling between transcription and mRNA maturation.coregulator ͉ Ewing sarcoma ͉ EWS-FLI1 ͉ polyadenylation ͉ splicing G ene expression in cancer cells is altered at the transcriptional level by many mutated oncogenes acting as transcriptional regulators. A second level of gene expression that is often altered in cancer cells is pre-mRNA splicing. Indeed, most human genes give rise to several transcripts with different exon content because of alternative splicing and alternative cleavage/ polyadenylation sites (1). Genes involved in major cellular programs often give rise to splice isoforms with distinct biological activities and deregulated expression in cancer (2, 3). In some cases, cancer-associated deregulation of alternative splicing arises from mutations within splicing regulatory sequences or from alterations of the expression of splicing factors involved in splicing regulation (2, 3). However, only few splicing factors have been found to be altered in cancer. Moreover, the role of another level of splicing regulation that involves transcriptional regulators has not been investigated yet.It is now widely accepted that pre-mRNA splicing and 3Ј-end maturation are tightly connected to transcription in Metazoans and that transcription impacts RNA processing (4, 5). It has been shown that the recruitment of processing factors and the maturation of pre-mRNAs occur at least in part cotranscriptionally and are enhanced by RNA polymerase II (Pol II) and its phosphorylation (...

A recently evolved class of alternative 3′-terminal exons involved in cell cycle regulation by topoisomerase inhibitors

“…The relative inability of DOXO to favour inclusion of cassette exons and internal ALEs (Fig. 1c,d) likely stems from its lack of inhibition of transcription elongation 23 , which mediates exon inclusion in response to CPT and UV 19,21,56 . Cassette-exon skipping and internal ALE repression are frequently induced by both DOXO and CPT 22 (Fig.…”

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

“…This suggests that YK-4-279 blocking of specific protein interactions from EWS-FLI1, such as RHA and p68, will alter the splicing program differently from the loss of EWS-FLI1. We also considered whether EWS-FLI1-modulated RNA pol II elongation activity was affected by YK-4-279 treatment, because this mechanism led to alternative poly(A) site selection for cyclin D1 (17). We did not note any alteration in polymerase elongation activity when stalled complexes were released; however, polymerase effects based on YK-4-279 treatment cannot be fully eliminated based on this limited investigation.…”

“…In addition, previous protein interaction studies identified the SF1 and U1C splicing factors as partners of EWS-FLI1 and discovered that EWS-FLI1 alters the splicing of an adenoviral transcript (14,15). Also, EWS-FLI1 has been shown to modify the IGFBP3 mRNA half-life (16) as well as directly slow the rate of RNA polymerase activity during cyclin D transcription, leading to a more oncogenic isoform, cyclin D1b (17). In total, these studies suggest functionally significant EWS-FLI1 activity in addition to transcriptional regulation driven by DNA binding (16).…”

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

“…Finally, it has also been reported that an oncogenic fusion between the multifunctional RBP EWS and the transcription factor FLI1, which is found in Ewing's sarcoma, can influence production of D1b. While EWS itself was shown to promote production of full-length D1a, the EWS-FLI1 fusion promoted production of D1b (Sanchez et al 2008). This was proposed to occur due to impaired transcription elongation in the presence of the fusion protein, which would allow more time for polyadenylation to occur in intron 4 before transcription of E5.…”