Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold

Abstract

Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.

Fig. 1. Sequence comparison of RNP-2 and RNP-1 motifs for PTB with the RRM family consensus (Kenan et al., 1991). The PTB motifs contain quite unusual amino acids relative to the consensus (indicated by shading). Positions of conserved hydrophobic positions are indicated by an asterisk.

Fig. 2. Superposition of 20 refined structures for PTB RRM-3 and PTB RRM-4 domains. The backbone traces for residues 334–433 in RRM-3 and 449–531 in RRM-4 are shown. The N- and C-termini are indicated for each domain and β-strands are numbered.

Fig. 3. Ribbon diagrams for PTB-34 and Sex-lethal. (A) Comparison of tandem domain structures of PTB and Sex-lethal. The relative orientation of the two domains shown for PTB is arbitrary, as is the structure of the inter-domain linker. The structure of Sex-lethal was solved crystallographically in the presence of bound RNA (Handa et al., 1999), which has been omitted from the figure. (B) Comparison of RRM-3 and RRM-4 domains from PTB with RRM-1 of Sex-lethal. PTB RRM-3 contains an additional strand (β5) on one side of the RNA binding surface. Note that the conformation shown for the β4–β5 loop is only one of many conformations that are consistent with the data (see Figure 2).

Fig. 4. Structure-based sequence alignment of RRM-3 and RRM-4 from PTB. Secondary structure elements are depicted above the sequences. Identical residues are shaded and the RNP-1 and RNP-2 motifs are boxed.

Fig. 5. Overview of protein and RNA constructs used in this study. (A) Schematic depiction of PTB constructs; RRM domains are indicated by shading. (B) Schematic diagram of the EMCV IRES; domain 1 is boxed.

Fig. 6. Binding curves for the interaction of full-length and truncated PTB constructs with EMCV IRES domain 1 RNA. Experimental details are given in Materials and methods. The data for each protein have been normalized. Analysis of such curves was used to determine dissociation constants for all the PTB constructs and mutants used in this study (Tables II and III).

Fig. 7. Mapping of sites of mutation onto the structure of PTB-34. The RNP-1 and RNP-2 motifs are indicated on the protein structure by dark shading. Large spheres (with underlined labels), positions of mutations that caused >1.5-fold decrease in binding; small spheres, positions of mutations that caused <1.5-fold decrease in binding (see Table II).

Fig. 8. Alignment of PTB and homologous sequences in the region of β4 and β5 of RRM-3, indicating the conservation of β5 in eukaryotes. DDBJ/EMBL/GenBank accession Nos for PTB and homologue sequences are: human (X62006), pig (X93009), rat (Q00438), C.elegans (Z36948), Arabidopsis thaliana (AF076924), NPTB (AF176085), ROD1 (AB023967) and hnRNP-L (X16135).

Fig. 9. The role of sequences flanking the core RRM domain in RNA recognition. (A) The structure of U2B” complexed with its cognate RNA (Price et al., 1998). For ease of comparison with the other structures only a portion of the stem–loop RNA (nucleotides 8–14) is shown and U2A′ (which does not interact with the portion of RNA included in the figure) has been omitted. The C-terminal helix is crucial for high-affinity RNA binding. (B) Structure of RRM-2 of PABP bound to poly(A) (nucleotides 1–7; Deo et al., 1999). The figure shows one RRM from the two-domain fragment that was present in the crystal. The N-terminal helical turn preceding β1 interacts specifically with the bound RNA. (C) Structure of RRM-2 of Sex-lethal bound to its cognate RNA (nucleotides 1–11; Handa et al., 1999). RRM-1, which lies on the right and also interacts with the RNA, has been omitted from the figure. (D) Superposition of the RNAs from U2B”, PABP and sex-lethal onto PTB RRM-3. The matrix required to superpose each protein domain on PTB RRM-3 was determined and applied to the RNA. In each case the conformation of the bound RNA clashes with the C-terminal end of the β4–β5 loop in PTB RRM-3 (indicated by arrows).