A nuclear protein affecting -actin mRNA localization | Gu et al. 43 Figure 2. Analysis of the proteins fractionated by RNA affinity chroma- tography. (A) SDS-PAGE analysis. Aliquots (30 l) of proteins in each purification step eluted from the RNA affinity chromatography were resolved in 10% SDS-PAGE. (Lane 1) Flow- through fraction. (Lanes 2–6) Proteins in five successive washing steps, respectively. (Lanes 7–9) Proteins in 0.5, 1.0, and 2.0 KCl elution fractions, respectively. Protein molecular mass is marked at right position. The small arrows denote the five proteins that have been microsequenced. (B) Gel shift assay using the affinity selected proteins. Aliquots (5 l) of each as in A (fraction) were incubated with 1 105 CPM of [32P]-labeled zipcode transcript and the complexes formed were resolved in 4% native gels. (C) Starting material. The arrows represent the RNA–protein complexes identified in Fig. 1 A. plexes M and S (the brain-enriched bands) contained a crosslinked protein with an estimated molecular mass of 92 kD. The migration difference of complexes M and S in native gel, and the identity in molecular mass on SDS- PAGE, suggested that complex S could be a dimer of com- plex M. From these data we conclude that the 68- and 92-kD proteins bind to the zipcode, the -actin mRNA lo- calization signal. The 68-kD band is likely ZBP1, but the other ZBP is preferentially expressed in brain. The 92-kD protein was named ZBP2. The fact that they segregate into separate complexes indicates that ZBP1 and ZBP2 do not bind simultaneously to the zipcode. Purification of ZBPs To identify the brain-enriched protein, we purified ZBPs by using RNA affinity selection techniques. We passed brain or cultured fibroblast extracts over an affinity chromatography column containing zipcode RNA. After extensive washes, the proteins retained on the column were eluted with increasing salt concentration steps and analyzed by SDS-PAGE with sil- ver staining (Fig. 2 A). Although the starting extract and flow-through fraction were heterogeneous (Fig. 2 A, lane 1), after a series of washes (lanes 2–5), specific proteins were eluted with increasing salt conditions (Fig. 2 A, lanes 7–9). The protein fraction eluted with 1 and 2 M KCl contained distinct protein bands (Fig. 2 A, lanes 8 and 9, arrows); three proteins were 68 kD and one was 92 kD. The estimated molecular masses were in close agreement with that of the UV-crosslinked RNA–protein complexes identified by SDS- PAGE (Fig. 1 B). A lower molecular mass protein (53 kD) had also been detected previously (Ross et al., 1997). To determine whether proteins eluted from the RNA af- finity column contained RNA binding activity, we per- formed the band mobility shift assay by incubating the ra- diolabeled zipcode with proteins from each of the purification steps (Fig. 2 B). In comparison with the starting material (Fig. 2 B, lane C), enhanced RNA binding activity was de- tected in 1- and 2-M KCl fractions (lanes 7 and 8). The RNA binding activity was not found in the flow-through (Fig. 2 B, lane 2), wash steps (Fig. 2 B, lanes 3–6), and 0.5-M KCl fraction (Fig. 2 B, lane 6), indicating that the RNA binding proteins were enriched in the 1- and 2-M KCl frac- tions after RNA affinity purification. Surprisingly, although the 1- and 2-M KCl fractions contained 92- and 68-kD pro- teins, after incubation with radiolabeled zipcode, only com- plexes M and S were formed (Fig. 2 B, lanes 8 and 9). The relative absence of complex F in the band shift assay sug- gested either that the 68-kD protein lost its RNA binding activity in the high salt elution condition, or that the bind- ing of the 68-kD protein to the zipcode needs additional proteins not present or active in the high salt fraction. Microsequencing of affinity purified proteins Proteins in the 2-M KCl fraction of RNA affinity column (Fig. 2 A, lane 9) were concentrated with a centricon-30 fil- ter, resolved in 12% SDS-PAGE, and visualized by Coo- massie blue staining. Four protein bands of 92, 70, 65, and 45 kD were eluted from the gel and microsequenced. Six peptides of the purified 92-kD protein, which we named ZBP2, were obtained after microsequencing. A database search with the peptide sequences of ZBP2 revealed that the six peptides matched a human nuclear protein, KSRP, that has been previously identified as a regulatory splicing factor (Min et al., 1997). The other three proteins were also identi- fied by their peptide sequences. The 70-kD protein was a homologue of a human protein, FBP, which was known as a transcription factor for the c-myc gene (Duncan et al., 1994). The 65-kD protein was an unknown protein. We did not sequence the 68-kD protein, as we assumed that this protein was ZBP1 (Ross et al., 1997). The 45-kD protein was iden- tified as ssDBF, a single-strand DNA binding factor in chicken (Smidt et al., 1995), and was homologous to a hu- man protein, ABBP, a type A/B hnRNP protein that plays a role in mRNA editing (Lau et al., 1997). Isolation of chicken cDNA for ZBP2 The cDNA-encoding ZBP2 was obtained by screening three chicken cDNA libraries and rapid amplification of cDNA ends (RACE)-PCR amplification of the 5 terminus. The ZBP2 and the human KSRP share 81% identity in the nucleic acid sequence and 86% identity in amino acid (aa) sequence (Fig. 3), indicating that ZBP2/KSRP is a highly conserved protein. As with human KSRP and ZBP1, ZBP2 also contains four hnRNP type K homology RNA binding (KH) domains and a COOH-terminal region enriched in glutamines, with four repeats of an AWEEYYK motif and an NH2-terminal proline-glycine rich domain (Fig. 3). How- ever, chicken ZBP2 contains a 47-aa segment before the first