Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons.

Splicing of fibroblast growth factor receptor 2 (FGFR2) alternative exons IIIb and IIIc is regulated by the auxiliary RNA cis-element ISE/ISS-3 that promotes splicing of exon IIIb and silencing of exon IIIc. Using RNA affinity chromatography, we have identified heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a splicing regulatory factor that binds to ISE/ISS-3 in a sequence-specific manner. Overexpression of hnRNP M promoted exon IIIc skipping in a cell line that normally includes it, and association of hnRNP M with ISE/ISS-3 was shown to contribute to this splicing regulatory function.

Identification of RNA-binding proteins that regulate FGFR2 splicing through the use of sensitive and specific dual color fluorescence minigene assays.

We have developed a series of fluorescent splicing reporter minigenes for the establishment of cell-based screens to identify splicing regulatory proteins. A key technical advance in the application of these reporters was the use of two different fluorescent proteins: EGFP and monomeric Red Fluorescent Protein (mRFP). Through establishment of stable cell lines expressing such dual color fluorescent reporters, these minigenes can be used to perform enhanced screens for splicing regulatory proteins.

Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing.

Alternative splicing of fibroblast growth factor receptor-2 (FGFR2) mutually exclusive exons IIIb and IIIc results in highly cell-type-specific expression of functionally distinct receptors, FGFR2-IIIb and FGFR2-IIIc. We previously identified an RNA cis-element, ISE/ISS-3, that enhanced exon IIIb splicing and silenced exon IIIc splicing. Here, we have performed comprehensive mutational analysis to define critical sequence motifs within this element that independently either enhance splicing of upstream exons or repress splicing of downstream exons.

A novel intronic cis element, ISE/ISS-3, regulates rat fibroblast growth factor receptor 2 splicing through activation of an upstream exon and repression of a downstream exon containing a noncanonical branch point sequence.

Mutually exclusive splicing of fibroblast growth factor receptor 2 (FGFR2) exons IIIb and IIIc yields two receptor isoforms, FGFR2-IIIb and -IIIc, with distinctly different ligand binding properties. Several RNA cis elements in the intron (intron 8) separating these exons have been described that are required for splicing regulation. Using a heterologous splicing reporter, we have identified a new regulatory element in this intron that confers cell-type-specific inclusion of an unrelated exon that mirrors its ability to promote cell-type-specific inclusion of exon IIIb.

A Non-sequence-specific double-stranded RNA structural element regulates splicing of two mutually exclusive exons of fibroblast growth factor receptor 2 (FGFR2).

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) mutually exclusive exons IIIb and IIIc represents a tightly regulated and functionally relevant example of post-transcriptional gene regulation. Rat prostate cancer DT3 and AT3 cell lines demonstrate exclusive selection of either exon IIIb or exon IIIc, respectively, and have been used to characterize regulatory FGFR2 RNA cis-elements that are required for splicing regulation. Two sequences termed ISE-2 and ISAR are located in the intron between exons IIIb and IIIc and are required for cell-type specific exon IIIb.