PWI motif-containing domains of SRm160 and hPRP3 bind RNA. (A) Domain organization of selected PWI-motif proteins from the SMART database (Schultz et al. 1998). The approximate location of PWI and RNA recognition motifs (RRMs), as well as regions of low sequence complexity (gray boxes) are indicated. (B) RNA EMSA of GST-SRm160(1–151) and GST proteins with T7-MCS RNA (see Materials and Methods). The amounts of each protein in the EMSA experiment are indicated in micromolars. A Coomassie-stained gel containing the purified proteins is shown in the left panel. (C) Supershift assay with GST-SRm160(1–151). Five micromolars of GST-SRm160(1–151) or 10 μM of GST was incubated with the T7-MCS RNA and increasing amounts of a polyclonal antiserum specific for GST. RNA binding was analyzed by EMSA. Note that the complexes formed specifically between GST-SRm160(1–151) and RNA were only separated for a short time, in order to observe a supershift with the anti-GST antibody. (D) RNA-EMSA with purified, baculovirus-expressed, wild-type SRm160 (SRm160-WT) and SRm160 lacking residues 1–151 (SRm160-ΔN1). Binding to the T7-MSC-RNA was assayed as in B and C. A Coomassie-stained gel containing the purified SRm160-WT and SRm160-ΔN1 proteins is shown in the left panel. (E) RNA-EMSA with GST-hPRP3(2–94) and the T7-MCS RNA. A Coomassie-stained gel containing the GST-hPRP3(2–94) protein is shown in the left panel.

A heterologous RNA-binding domain can functionally substitute for the PWI domain of SRm160. (A) Diagram of pre-mRNA reporters and test proteins. Pre-mRNA reporters were derived from exons 3 and 4 of the Drosophila doublesex (dsx) gene and contained a deletion in the 5′-splice site to eliminate splicing activity. One of the reporters contains three tandem RNA-binding sites for the phage MS2 coat protein in exon 4, as indicated. The position of the RNase protection probes used to monitor 3′-end cleavage of transcripts from each reporter is indicated. Test proteins corresponding to SRm160-WT and SRm160-ΔN1 (Fig. 1D legend), with or without an MS2 RNA-binding domain fused to the N terminus, were analyzed. All four proteins contained an N-terminal Flag-epitope for detection. (B) Immunoblots of proteins from cells transfected with or without expression plasmids for the four test proteins shown in A. The immunoblot in the top panel was probed with the anti-MS2 antibody, and the immunoblot in the bottom panel was probed with the anti-Flag antibody. Note: Both MS2 fusion proteins were detected with the anti-Flag antibody after prolonged exposure (data not shown). (C) RNase protection analysis of reporter transcripts with the 3′-end protection probes illustrated in panel A. Cells transfected with each reporter and test protein combination are indicated. A pol III reporter (pSPVA) was cotransfected in each case as an internal control for transfection efficiency and RNA recovery. RNase protections were performed on RNA isolated from both nuclear (N) and cytoplasmic (C) fractions from transfected cells, as indicated. Ratios of cleaved to uncleaved RNA in each fraction are indicated in the bar graphs with standard deviations, as determined from three separate analyses. Note that the two gel panels are from the same experiment and were separated only to facilitate labeling the positions of the different size RNA products.

Solution structure of the PWI motif from SRm160. (A) Best-fit superimposition of the backbone atoms from the 20 lowest-energy structures of SRm160 residues 27–126. Residues 127–134, which are disordered, are excluded from the figure for clarity. Helical regions are colored, and nonhelical regions are in gray. Locations of the signature Pro (50), Trp (51), and Ile (52) residues, as well as the highly conserved Phe (101) residue, are indicated in black. (B) Ribbon representation of the lowest-energy calculated structure. The orientation and color scheme are the same as in A. (C) Sequence of the polypeptide used in structure determination. Residues labeled in brown correspond to those that constitute the PWI domain, and the black letters indicate those found in the flanking sequences. Colored cylinders above the sequence indicate α-helices, and follow the same color scheme used in A and B.

Surface renderings of the PWI motif structure. (A) Electrostatic surface potential map of SRm160(27–126). Red represents regions of negative charge, blue shows positive charges, and white is neutral. The molecule on the left is in the same orientation as in Figure 3, and the molecule on the right is rotated by 180° about the indicated axis. (B) Map of the residues in SRm160(18–134) that have an altered amide chemical shift upon binding dsDNA (magenta). White surfaces correspond to residues that do not have altered chemical shifts, or ambiguous results due to spectral overlap.

Cooperative roles of core PWI motifs and conserved, adjacent, basic domains in RNA binding. (A) Schematic diagram of GST-fusion proteins used in RNA-EMSA experiments shown in B–F. Regions corresponding to the conserved PWI motifs in the SRm160 and PRP3 constructs are indicated by the hatched boxes. The structured region of the p73 SAM domain is represented by the checkered box. The residue numbering for SRm160 and the construct used in structure determination, including secondary structure, is depicted above the other constructs. Approximate locations of the three lysine-to-alanine substitutions (Lys 20, Lys 22, and Lys 23) in the GST-SRm160(18–134)-TM (“Tail Mutant”) protein are indicated by small triangles. (B) Coomassie-stained gel of GST-fusion proteins used for gel shift analysis. Masses of the molecular weight markers are indicated. (C) Simultaneous mutation of Lys 20, Lys 22, and Lys 23 to alanines in the GST-SRm160(18–134) protein leads to an abrogation of RNA binding (cf. lanes 9–14 and 3–8). (D) The N-terminal basic domain is required but not sufficient for RNA-binding activity of the PWI motif. Whereas the GST-fusion protein that consists of residues 18–134 binds to the T7-MCS RNA (see Materials and Methods) in the same manner as the N-terminal fragment used in Figure 1 (lanes 3–8), subfragments 18–26 and 27–134, in which the structure of the PWI domain is maintained, are not able to bind RNA in a similar manner. Interactions of the 18–26 fragment (lanes 9–14) with RNA are reduced, and the 27–134 fragment does not appear to interact at all. Similar results were observed in all cases for dsDNA (data not shown). GST itself does not shift the RNA (lane 2). (E) The N-terminal basic region specifically cooperates with the PWI domain to bind RNA. Substitution of the PWI motif with the SAM domain from p73, another helical bundle protein with a similar pI (lanes 15–20), does not rescue binding activity provided by the PWI motif (lanes 3–8) and results in a similar level of binding as observed for the basic region alone (cf. D, lanes 9–14). The SAM domain on its own also does bind RNA (lanes 9–14). (F) Efficient RNA binding of the hPRP3 PWI motif requires a C-terminal basic domain. Removal of 17 amino acid basic sequence from the C terminus of hPRP3 (lanes 3–8) leads to a considerable reduction in binding relative to the GST-PRP3(1–93) fusion protein (lanes 9–14).

Multiple alignment of PWI motifs and conserved adjacent basic domains. A representative set of 11 PWI motif proteins are shown aligned. The set represents proteins with either an N-terminal PWI motif (top five sequences) or a C-terminal PWI motif (bottom five sequences). Highly conserved residues among all PWI motifs are colored in red. Residues that are specifically conserved in proteins containing an N-terminal motif are indicated in yellow, and residues specifically conserved in proteins with a C-terminal PWI motif are indicated in blue. Only residues that are similar in at least four out of five of the grouped sequences are colored. Basic amino acids located in the flanking regions are in bold type. Note that hPRP3, which lacks an N-terminal basic region, has a disproportionately higher number of basic residues C-terminal to the PWI motif. The region corresponding to the core PWI motif is boxed, and the secondary structure of the corresponding regions in the SRm160 motif is indicated by cylinders above the sequences.