G·A·B·B·A

Alternative splicing can generate different mRNAs from a single gene, and is the major contributor to protein diversity in higher eukaryotes. Regulation of alternative splicing occurs mainly through the action of trans-acting factors that bind to cis-acting sequences, exerting positive or negative influences on the assembly of the spliceosome. Polypyrimidine Tract Binding protein (PTB/hnRNPI) is a negative splicing regulator containing four RNA recognition motifs (RRM). The Tpm1 exon 3 is repressed in smooth-muscle and PTB, despite being widely expressed, 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, as defined from artificial tethering assays using the bacteriophage MS2 coat protein. This domain reconstitutes splicing repression in a tissue specific manner and is dependent on the other regulatory elements. 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 to understand its mechanism. A search for PTB co-regulators that bind the same surface as Raver1 does was carried out and 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 in vitro system was set out to map the RRM2 binding site when tethered, which revealed it can bind to the P3 site directly, possibly 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.