| Summary: | Polypyrimidine Tract Binding protein (PTB/hnRNPI) is a negative splicing regulator containing four RNA recognition motifs (RRM). The <i>Tpm1</i> exon 3 is repressed in smooth-muscle and PTB is a regulator of this splicing event. Repression of this exon is dependent on flanking PTB binding sites in the intron, the upstream site overlapping with the polypyrimidine tract (P3). The PTB minimal repressor domain consists of RRM2 with the following interdomain linker. Raver1 is a PTB co-repressor in this system, and it binds to RRM2, on the surface opposite the RNA binding one. The components of the PTB minimal repressor domain, RRM2 and the linker region have been studied. A search for PTB co-regulators that bind the same surface as Raver1 resulted in the identification of several PTB binding proteins with diverse functions. The role of the RNA and Raver1 interaction surfaces of RRM2 was analysed by mutagenesis of key residues. Impairment of the Raver1 interaction surface caused a decrease in activity, and also impaired binding to other proteins. Surprisingly, RNA binding by RRM2 was required for PTB repressor activity, even when tethered via MS2, indicating it can bind to two different RNA sites simultaneously. An <i>in vitro</i> system was set out to map the RRM2 binding site when tethered, which revealed it can bind to the P3 site directly, causing the RNA to loop out. This simultaneous binding can be competed off with rPTB, resulting in a reduction in exon skipping levels. My data supports models of PTB mediated repression by inducing RNA looping of the exon and flanking introns.
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