HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition

Serena Bonomi 2 Anna di Matteo 2 Emanuele Buratti 1 Daphne S. Cabianca 0 Francisco E. Baralle 1 Claudia Ghigna 2 Giuseppe Biamonti 2 0 Division of Regenerative Medicine, Stem Cells , and Gene Therapy, Dulbecco Telethon Institute at San Raffaele ScientiEc Institute , 20132 Milan, Italy 1 International Centre for Genetic Engineering and Biotechnology , 34012 Trieste, Italy 2 Istituto di Genetica Molecolare , Consiglio Nazionale delle Ricerche (IGM-CNR), 27100 Pavia, Italy *To whom correspondence should be addressed. Tel: +39 382 546324; Fax: +39 382 422286; Email: Correspondence may also be addressed to Giuseppe Biamonti. Tel: +39 382 546322; Fax: +39 382 422286; Email: The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. - Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. "Ron, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of "Ron and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of "Ron, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote "Ron production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies. Alternative splicing is a mechanism of gene expression regulation that either modulates the production of protein isoforms with distinct structural and functional properties or affects mRNA stability, through the introduction of premature stop codons, and translatability, by removing targets sites for microRNAs. Its prevalence in regulatory circuits is proven by the fact that >90% of human genes encode transcripts that undergo at least one alternative splicing event with a frequency higher that 10% (1,2). Moreover, alternative splicing contributes to the appropriate spatio-temporal regulation of cellular and developmental processes and to the response to a wide range of extracellular stimuli (3). A detailed molecular analysis has revealed that alternative splicing decisions involve regulatory sequences, located both in exons and in the flanking introns, which promote (enhancers) or inhibit (silencers) the recognition of splice sites. These elements comprise the target sequences for RNA-binding proteins most of which belong to two groups of widely expressed antagonistic splicing regulatory factors: the SR (serinearginine-rich) factors that usually promote exon recognition and the group of hnRNP (heterogeneous nuclear ribonucleoprotein) proteins, which in general play an inhibitory role (4). Notably, during tumor progression, stimuli from the tumor microenvironment may affect the expression and/ or activity of splicing regulatory factors thus perturbing the physiological splicing program of genes involved in all major aspects of cancer cell biology, including cell cycle control, proliferation, differentiation, signal transduction pathways, cell death, angiogenesis, invasion, motility and metastasis (46). In many cases, unscheduled activation of splicing programs typical of embryonic development may occur. However, completely uncharacterized new splicing isoforms are frequently generated as well (7). An increasing body of evidence indicates that splicing variants of many cancer-related genes can directly contribute to the oncogenic phenotype and to the acquisition of resistance to therapeutic treatments (46). Hence, understanding the functional role(s) of cancer-associated alternative splicing variants and the mechanisms underlying their production offers the potential to develop novel diagnostic, prognostic and more specific anticancer therapies. We have contributed to this topic by unveiling the connection between the expression level of splicing factor SRSF1 and the behavior of tumor cells (8). We have shown that SRSF1 (a member of the SR family also known as SF2/ASF) can regulate the epithelial-to-mesenchymal transition (EMT) and the migratory properties of cancer cells (8). EMT is a complex gene expression program through which terminally differentiated epithelial cells acquire mesenchymal features including the ability to efficiently move as single cells through the extracellular matrix (9,10). The EMT program is physiologically important during embryogenesis when it is crucial for organogenesis. However, in adults EMT occurs only during wound healing or it is involved in the metastatic spreading of epithelial cancers (9,10). SRSF1 is an oncoprotein upregulated in many human tumors (11). The involvement of SRSF1 in the EMT program derives from its ability to affect the splicing program of the tyrosine kinase receptor and proto-oncogene Ron. We have shown that SRSF1 promotes the production of Ron, a constitutively active isoform, through skipping of exon 11. More specifically, SRSF1 acts by directly binding to an exonic splicing enhancer (ESE) located in the constitutive exon 12 (8). As expected, additional factors such as hnRNP H and A2/B1 have a role in controlling splicing of Ron exon 11 and production of DRon. Recently, hnRNP H has been shown to promote the expression of DRon by binding to an exonic splicing silencer (ESS) in exon 11, while the mechanism of action of hnRNP A2/B1 has not yet been characterized (12,13). We have previously shown that the activity of the ESE bound by SRSF1 is counteracted by an ESS located upstream of ESE in the same Ron exon 12 (8). However, the molecular mechanism underlying the ability of the ESS to prevent skipping of exon 11 is still unknown. In this manuscript, we report the characterization of the ESS element in exon 12 of the Ron gene. We show that the ESS is bound by hnRNP A1, a known antagonist of SRSF1 activity in splicing decisions (14). Intriguingly, binding of hnRNP A1 to the ESS sequence in vitro prevents the interaction of SRSF1 to the downstream ESE, which can be relevant for its ability to promote Ron exon 1 (...truncated)