The organization of RNA contacts by PTB for regulation of FAS splicing.
Bottom Line: RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA.The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker.Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.
Affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK.Show MeSH
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Mentions: The preceding data indicated the importance of RNA contacts by RRM3 and 4 for PTB repressor activity, despite the low specificity of RRM4 contacts. RRM3 and 4 form a stable di-domain with back-to-back packing of the two RRMs involving the highly conserved linker (11,40). Mutation of the packing interface decreased affinity of recombinant RRM34 di-domain for RNA, and reduced repressor activity of full length PTB upon the CSRC N1 exon in a co-transfection assay (13). We therefore decided to test the packing mutant, C4pack (E502K; V505E; I509K, all in helix 2 of RRM4) for its effects upon FAS splicing (Figure 6A). The packing mutant showed a substantial reduction in repressor activity, with 46% of WT activity at 30 ng/μl (Figure 6A lanes 6, 7), while the RRM4 binding mutant had 39% of WT activity in this experiment (Figure 6A, lanes 8, 9). We tested the effects of the packing mutation upon RNA contacts by RRMs 3 and 4 by tethered OH radical probing. All contacts by RRMs 3 and 4 were abolished by the packing mutant (Figure 6B), indicating that stable RNA contacts by both RRMs are dependent upon their back-to-back packing. In contrast, no deleterious effects of the packing mutation were observed in a filter binding assay (Table 1).
Affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Tennis Court Road, Cambridge, CB2 1QW, UK.