Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3’ Splice Site Recognition

Eperon IC (2013) Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3' Splice Site Recognition. PLoS ONE 8(10): e77208. doi:10.1371/journal.pone.0077208 Editor: Emanuele Buratti Intron Retention in the Alternatively Spliced Region of RON Results from Weak 3' Splice Site Recognition Lindsay D. Smith 0 Christian M. Lucas 0 Ian C. Eperon 0 0 Department of Biochemistry, University of Leicester , Leicester , United Kingdom The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3' splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5' of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo. - RON is a universally expressed tyrosine kinase receptor for macrophage stimulating protein (MSP) [1]. MSP-dependent activation of RON induces downstream signalling pathways involved in wound healing, liver regeneration, bone resorption, embryogenesis and the immune response [2-7]. There are eight known variants of RON including 170, 165, 160, 155, 110, 55, 85 and 90, all of which arise through splicing alterations [8]. Some RON isoforms, such as RON 165, 160 and 155, are constitutively active. As with overexpression, constitutive activation confers an invasive and motile phenotype on epithelial cells and promotes metastasis (EMT) [9-13]. In particular, it results in the break-down of cellcell contacts, promoting cell mobility and matrix invasion [14,15] and the progression of tumours [8,16]. Inappropriate RON signalling has been implicated in pancreatic [17], brain [16], colorectal [18], mammary [19], gastric [18], ovarian [20], hepatocellular [21], prostate [22], urinary [23] and renal [24] carcinomas. RON 165 mRNA has been identified in breast and gastric carcinoma tumours and has been directly linked to the development of an invasive phenotype in these tissues [25,26]. This isoform maintains the reading frame but lacks exon 11, which codes for the 49-amino acid trans-membrane region of the receptor (FL; Figure 1A). The loss of exon 11 results in incorrect processing of the receptor and localization to the cell cytoplasm. Homodimerization then occurs between the mislocalized receptors, due to incorrect disulphide bond formation, which results in constitutive activation [12,25,27]. Skipping of exon 11 and expression of RON 165 is stimulated by the binding of SRSF1 to RON exon 12 [19]. This is likely to be one route that mediates the known protooncogenic activity of SRSF1 [28]. Exon 11 skipping, SRSF1 expression and cell motility are regulated through phosphorylation of Sam68, which is a target of the ERK1/2 signalling pathway. The ERK signalling pathway is often itself miss-regulated in cancer and is, in addition, a signalling pathway downstream of RON [29,30]. Skipping of exon 11 is also stimulated by hnRNP H, which interacts with the 5 end of exon 11 and correspondingly promotes cell invasiveness [31]. One of the striking features of splicing between exons 10, 11 and 12 of RON is that a variable but often high proportion of the RNA remains unspliced in both normal tissues and tumours [19]. Similarly high levels were seen when minigenes containing the genomic sequences from exon 10 to exon 12 were expressed in cell lines [19,31]. Indeed, unspliced RNA was by far the predominant product of one such minigene, and Figure 1. RON splicing and splicing complex formation in vitro. (A) Diagrams of RON pre-mRNA substrates and their expected patterns of splicing. Boxes represent RON exons 10 (210 nt), 11 (147 nt) and 12 (166 nt), and lines represent introns 10 (87 nt) and 11 (80 nt) respectively. (B) Time courses of splicing in HeLa nuclear extract of the single intron substrates. Pre-mRNA substrates 10-11 and 10-11 begin at the 5 end of exon 10, with an additional GGG for transcription initiation, and end at the 3 end of exon 11 plus a 7 nt intron portion of either wild-type exon 11 5 splice site sequence (11,12) or consensus 5 ss sequence (GUAAGUU) (10-11). Pre-mRNA substrates 11-12 and 11-12 begin at nt 7 of exon 11, which forms a natural GGG tract, and end either at the end of exon 12 (11,12) plus 7 nt of the wild type 5 splice site or with an additional 3 consensus 5ss (11-12). G premRNA is derived from -globin exons 2 (226 nt) and part of exon 3 (56 nt), with a reduced intron (106 nt). DNA marker lengths are as shown (nt). (C) Analysis by native gel electrophoresis of pre-mRNA-containing complexes following incubation under conditions permitting assembly of complex E for the times shown. Nuclear extract was pre-incubated where indicated (U1 KD) with a 2-Omethyl oligonucleotide complementary to the 5 end of U1 snRNA prior to addition of pre-mRNA or incubated with heparin following assembly. Spliceosomal complexes E and H are labeled to the left of the panel. (D) Native gel electrophoresis after incubation of pre-mRNA in nuclear extract under conditions permitting spliceosome assembly. Complex H and the spliceosomal complexes A, B, C are labeled to the left of the panel. doi: 10.1371/journal.pone.0077208.g001 it remained so even when exon 11 inclusion was stimulated at the expense of skipping by knockdown of hnRNP H [31]. Such a preponderance of unspliced RNA is remarkable. One possible explanation is that splicing of RON pre-mRNA is suppressed specifically, as a mechanism for modulating the levels of expression of RON. The purpose of this research was to identify putative sites of such regulation and the mechanisms involved. Materials and Methods Construct synthesis Mutants were constructed from overlapping PCR reactions done with Phusion DNA high fidelity DNA polymerase (Thermo Scientific) and cloned, after digestion with DpnI, into pCRBlunt II-TOPO vector (Invitrogen). Following sequence confirmation, constructs were amplified for transcription by PCR. In each case the transcription template was generated using a forward primer containing a T7 polymerase promoter sequence, GGG to provide an efficient initiation site for transcription and a sequence complem (...truncated)