We have demonstrated that PK-M
ME splicing involves a two-component circuit: exon 9 is repressed and exon 10 is activated in proliferating cells, and these two effects are essentially independent (Supplementary Figure S6
By duplicating and swapping ME exons in a PK-M minigene, we showed that the key cis-elements controlling PK-M alternative splicing are located within the ME exons themselves. This is the first demonstration of exon-centric alternative splicing regulation via ME exons. As a first proof of this principle, we mapped a bona fide SRSF3 ESE in exon 10 that proved sufficient to activate exon 9 splicing in cancer cells when placed in this exon. Although double inclusion of exon 10 was not the major product from the exon 10-duplication minigene (Figure ), this cannot be due to repression via the flanking intron 8 and 9 regions, because exon 10 was spliced efficiently when it was cleanly swapped with exon 9. Instead, we speculate that intronic elements are likely involved in the ME exon selection properties of PK-M.
Remarkably, the SRSF3 ESE motif in exon 10 differs from the corresponding exon 9 sequence by only two nucleotides, which are wobble bases in the respective codons (Supplementary Figure S3
A and B). The first wobble base is highly conserved, perhaps reflecting the importance of this nucleotide in mediating SRSF3 recruitment and functionality. The corresponding two nucleotides in exon 9 are also conserved, suggesting selection against the creation of an exon 9 SRSF3 activation motif (Supplementary Figure S3
B). However, we cannot rule out the existence of a corresponding exonic splicing silencer element in exon 9. The use of two wobble nucleotides to code for a key splicing signal illustrates the impact of sequence changes that would be translationally neutral, except that they drastically affect the structure of the resulting protein by changing alternative splicing of the entire exon (Cartegni et al., 2002
Although mutating just two nucleotides in exon 10 to the corresponding exon 9 sequence (9 SR minigene) gave rise to more double-skipped and PK-M1 mRNAs, this was not the case when the surrounding exon 9 sequence was also duplicated (9 1530 and 9 30 minigenes). This probably reflects the presence of additional cis-elements in exon 9, such as putative ESEs that may compensate for the loss of exon definition upon mutation of the SRSF3 motif.
Whereas the 5′ss of exons 9 and 10 do not play a dominant role in exon selection—considering that the M1
mRNA levels do not change upon swapping these 5′ss (Supplementary Figure S2
B)—mutational analysis indicates that the 3′ss are necessary for definition of their respective exons, and they compete with each other. Exon definition, and ultimately, proper ME exon selection in the PK-M
gene, is apparently dependent on the outcome of competition between the alternative 3′ss. We speculate that the recovery of endogenous PK-M1
transcripts upon SRSF3 knockdown may reflect this competition mechanism, resulting in a loss of exon 10 definition, and allowing the basal splicing machinery to be recruited to the exon 9 3′ss. Loss of exon 10 definition is also supported by the increase in double-skipped RNA from the wild-type minigene upon SRSF3 knockdown. However, it is unclear why the extent of PK-M1
inclusion is weaker in minigene than in endogenous RNA, leading to the accumulation of unproductively spliced double-skipped RNA.
We were surprised to find no rescue of exon 9 inclusion when its 5′ss was swapped with that of exon 10—despite a report that hnRNPA1 represses exon 9 inclusion by binding to the exon 9 5′ss (David et al., 2010
)—as this swap presumably removed the repressive hnRNPA1 binding site; perhaps this lack of rescue reflects contextual effects. However, our results confirm and extend an earlier study that duplicated the exon 10 5′ss in a heterologous minigene reporter, and found no change in PK-M
splicing (Takenaka et al., 1996
). Moreover, in the context of intact pre-mRNAs, it is not known how well hnRNPA1 binding to a motif that is part of a 5′ss can compete with binding of spliceosomal components, such as U1 and U6 snRNPs. Given that hnRNPA1 does affect exon 9 inclusion in vivo
(Clower et al., 2010
; David et al., 2010
), hnRNPA1-induced exon 9 repression could occur either indirectly—through regulation of a splicing factor that in turn regulates PK-M
alternative splicing—or through additional cis
-elements located elsewhere on the PK-M
The change in endogenous PK-M1
upon SRSF3 knockdown was roughly comparable to the effects of knocking down the known repressors of exon 9, hnRNPA1/A2 and PTB (Clower et al., 2010
; David et al., 2010
). However, knocking down these factors did not completely rescue exon 9 inclusion, and as in other systems, we anticipate the existence of additional activators of exon 10 and/or repressors of exon 9 that contribute to maintaining exon 10 definition in proliferating cells. With respect to the enhancer region we identified in exon 10, knockdown of other candidate splicing factors identified by RNA-affinity chromatography—hnRNPK and RBM3—did not change PK-M
splicing ratios (data not shown).
Consistent with its expected ability to facilitate cellular proliferation by altering glycolytic metabolism, SRSF3 is overexpressed in ovarian cancers (He et al., 2004
) and cervical cancer cell lines, whereas in normal cervical tissue, its expression is restricted to the basal proliferating layers (Jia et al., 2009
). SRSF3 is also a target of the oncogenic β-catenin/TCF-4 pathway in colorectal cancer cells (Goncalves et al., 2008
). Moreover, SRSF3 overexpression is sufficient for transformation of immortal mouse fibroblasts (Jia et al., 2010
), indicating that similar to its paralog, SRSF1 (Karni et al., 2008
), SRSF3 is an oncoprotein. Given the multitude of potential SRSF3 downstream target genes, we expect that SRSF3-mediated tumorigenesis reflects splicing changes in multiple effector genes, including PK-M
Important unanswered questions include: what are the additional factors that govern exon 9 and 10 use in tumor cells? Can the PK-M2 isoform be completely switched to the PK-M1 isoform by manipulating splicing factor levels in tumor cells? How is exon 9 preferentially selected in quiescent cells? Are there tissue-specific differences in the mechanisms of exon 9 selection in differentiated cells? Answers to these questions could provide the basis to develop splicing-targeted cancer therapeutics.