A Novel Set of Nuclear Localization Signals Determine Distributions of the αCP RNA-Binding Proteins

Posttranscriptional controls play a major role in the regulation of eukaryotic gene expression (24, 65). These controls (i) can increase the complexity of nuclear RNAs via alternative splicing and editing, (ii) can modulate information flow from the nucleus to cytoplasm, and (iii) can alter levels and sites of protein synthesis via controls over mRNA stability, translation efficiency, and subcellular localization (4, 56, 68). RNA-binding proteins that mediate these controls can be categorized based on the presence of one or more conserved RNA-binding motifs (for reviews, see references 6 and 37). The sequence specificity of these proteins, the identities of their RNA targets, and the respective mechanisms of action are therefore of significant interest. Studies from our laboratory and others have focused on the structures and actions of a subfamily of RNA-binding proteins, the αCPs (31, 39). These proteins, also referred to as PCBPs (17) and hnRNP Es (34, 58), contain a triplication of the KH domain (43, 69). The 70-amino-acid KH domain comprises a triple-β-sheet platform supporting three α-helical segments (35, 36, 50, 51). Cocrystal structures reveal that the KH domain can interact in a highly specific manner with four to five contiguous bases in a target RNA (5, 27). Two KH domain subtypes have been identified: the type 1 KH domain (e.g., KH3 of hnRNP K) has a C-terminal βα extension, and the type 2 KH domain (e.g., ribosomal protein S3) contains an N-terminal αβ extension (21). The KH domains in the αCPs are type 1 (40). KH domains are often represented in proteins in multiple copies. Since each KH domain has the potential to independently interact with a target RNA sequence, the complexity and specificity of RNA interaction for these proteins can be quite high (66, 74; our unpublished data). Our laboratory has focused on the role of αCPs in mRNA stabilization. These studies have defined a cytosine (C)-rich cis-acting stability element within the 3′ untranslated region (UTR) of the human (h) α-globin mRNA that serves as a binding site for αCP both in vitro and in vivo (28, 32, 83). Studies in our laboratory and others have identified closely related αCP binding sites in the 3′ UTRs of additional highly stable mRNAs (7, 10, 25, 61, 72, 86). In the case of the hα-globin mRNA, the RNP “α-complex” in the 3′ UTR appears to represent a 1:1 interaction of αCP with the C-rich motif (8), although there is evidence that additional proteins may also bind at this site (30, 63, 84, 85). These data suggest that the α-complex may represent a widely distributed and general determinant of mRNA stabilization (25). The αCPs involved in α-complex assembly can represent several isoforms (8, 31, 39). Whether these various αCP isoforms are performing unique functions or are redundant in their actions is not known. The functions mediated by αCP RNP complexes are in fact quite diverse (for review, see reference 41). As mentioned above, assembly of the 3′ UTR α-complex in α-globin mRNA mediates mRNA stabilization (32, 82, 83). This stabilization may reflect direct steric protection of the 3′ UTR from endonucleolytic attack (79), and/or it may reflect protection of the poly(A) tail from rate-limiting decay via interactions in cis between the bound αCP and the poly(A)-binding protein (30, 49, 78). αCPs also mediate translational controls. An array of αCP binding sites within the 3′ UTR of the 15-lipoxygenase mRNA has been linked to developmentally regulated translational repression during erythroid maturation (56-58). In contrast, association of αCP with the 5′ UTR of the polio viral RNA serves as an enhancer of internal ribosome entry site-mediated translation (2, 3). αCP binding within the 3′ UTR has also been implicated in the activation of maternal mRNA translation in early embryonic development in Xenopus via control of cytoplasmic polyadenylation (59). Additional systems are reported to involve αCP binding in the control of various aspects of mRNA expression (63, 84, 85; reviewed in reference 41). Thus, the targets and actions of the αCPs are quite diverse and may reflect the actions of one or more of the defined αCP isoforms. αCP isoforms are encoded by four unlinked loci in the human and mouse genomes: PCBP1, PCBP2, PCBP3, and PCBP4 (38, 39, 75) (see also Fig. ​Fig.1,1, left). Each locus has been mapped, sequenced, and characterized for mRNA structure (38, 39). A total of five major αCP isoforms have been identified in human or mouse tissues: αCP1, αCP2, αCP3, αCP4, and a major αCP2 splice variant, αCP2-KL, that differs from αCP2 by the exclusion of a 31-amino-acid segment in the region between the KH2 and KH3 encoded by a single exon (exon 8a) (17, 38, 39). These proteins are broadly expressed in human and mouse tissues and demonstrate polyC-binding specificity (34, 38, 39; unpublished observations). αCP1 and αCP2 share the highest level of amino acid sequence similarity at 89% (75), αCP3 is more divergent, and αCP4 is the most distantly related (52% divergence from αCP2 [39]). Each protein contains three similarly spaced KH domains; two KH repeats are located in the N terminus followed by a nonconserved region of variable length, and the third KH domain is located at the C terminus. Posttranslational modifications may regulate the binding of αCPs to RNA. For example, phosphorylation of αCP1 and αCP2 results in a marked decrease in RNA-binding activity (34). An additional major determinant of αCP isoform function may relate to subcellular localization. Although the defined roles of αCP in mRNA stability and translational control suggest a cytoplasmic localization, prior attempts to sublocalize αCPs did not resolve the question of whether αCP is in fact nuclear or cytoplasmic (17, 18). FIG. 1. αCP isoforms and detection of endogenous αCP1, αCP2, and αCP2-KL. The five major αCP isoforms are shown on the left. The corresponding genetic loci and protein designations are noted. The position of each KH domain ... Protein transport from cytoplasm to nucleus is mediated by multiple families of soluble factors. It is generally accepted that nuclear proteins carry a nuclear localization signal (NLS) (11, 45, 53, 67). NLSs vary considerably from relatively short stretches of residues to large protein domains with relaxed sequence conservation. Although different in structure, NLSs all seem to play the same role: recognition by soluble factors that mediate transport through the nuclear pore complexes (19, 20, 42, 54, 55, 77). In the present study each of the five major isoforms was individually assessed for subcellular localization, and the signals underlying their nuclear versus cytoplasmic distribution were defined. These data demonstrate an unexpected complexity in αCP isoform compartmentalization based on a novel set of nuclear localization motifs.