La-Related Protein 4 Binds Poly(A), Interacts with the Poly(A)-Binding Protein MLLE Domain via a Variant PAM2w Motif, and Can Promote mRNA Stability

The RNA binding domain of the conserved La protein consists of a La motif (LaM) and an RNA recognition motif (RRM) that work together to recognize UUU-3′OH on small nascent transcripts and to protect them from 3′ exonucleases (7, 45). In addition to this, La proteins can modulate mRNA translation (30, 63-65). The LaM-RRM arrangement has been found in La-related proteins 1 (LARP1), 1b, 4, 4b, 6, and 7, which have been separately conserved during evolution (8, 10) (LARP4b is also referred to as LARP5 in multiple databases and here will be designated LARP5/4b). LARP7 is specific for 7SK snRNA, which it recognizes in part via UUU-3′OH (29, 46). LARP6 binds to a stem-loop in the 5′ untranslated regions (UTRs) of collagen mRNAs in a uracil-dependent manner (15), and LARP1 was shown to bind poly(U) and to a lesser extent poly(G), but not poly(A) or poly(C) (51). Consistent with these specificities, LARP1, -6, and -7 have conserved all of the amino acids involved in UUU-3′OH recognition in La-RNA crystals (37, 66), while LARP4 and -5/4b have diverged, suggesting alternative RNA binding (8). Moreover, an invariant divergence in all of the LARP4 and -5/4b sequences available occurs in a most critical residue involved in base-specific recognition seen in La-RNA crystals, corresponding to human La Q20, suggesting a conserved difference in RNA recognition (8). Although the LaM-RRM in La protein recognizes RNA in a unique way (8, 45), whether LARPs share this or have adopted alternative modes of RNA recognition is unknown. Of the LARP families studied for function, LARP1, -5/4b, and -6 appear to be involved in mRNA metabolism and/or translation (9, 14, 15, 51, 57). Of these, LARP1 and -5/4b interact with poly(A) binding protein (PABP), although the precise mechanisms were not reported (9, 14, 57), whereas LARP6 appears to block assembly of its associated mRNAs with initiating ribosomes (15). Translation is facilitated by interactions of the 5′ cap and 3′ poly(A) of the mRNA by eukaryotic initiation factor 4E (eIF4E) and PABP (47). The translation initiation activity of PABP can be regulated by PABP-interacting protein 1 (Paip1), which stabilizes initiation complexes via interactions with the 40S ribosome (18, 47). Multiple molecules of PABP can bind poly(A) tails (5, 44), and some data suggest that at least two molecules of poly(A)-associated PABP are required for efficient translation initiation (2). PABP can engage a variety of protein partners via their common PABP interaction motif 2 (PAM2) sequences, including Paip1, Paip2, eRF3, GW182 (TNRC6C), ataxin 2, Tob2, and poly(A) nuclease, representing different mechanisms of control involving translation initiation and termination, as well as mRNA stability (19, 33, 34, 41, 44, 54, 60, 69). Since the PAM2 motifs make direct contacts with the MLLE domain of PABP (39, 40), proper signal integration presumably involves their competition for PABP (25). A model in which the PAM2 motifs of eRF3 and poly(A) nucleases compete for PABP reflects a balance of translation termination and mRNA deadenylation activities (25, 39, 41, 55). In rat neurogenic cells, LARP5/4b (KIAA0217) was a component of an mRNA-protein (mRNP) complex associated with PABP that could bind poly(A) in a Northwestern blotting assay, although no other RNAs were tested (3). A recent report demonstrated that two broad regions of LARP5/4b interact with PABP to stimulate translation, although RNA binding was not examined (57). While human LARP4 and -5/4b are most homologous in their LaM-RRMs, they share patchy homology in the ∼500 amino acids outside this region (8). Here, we identify variant PAM2 motifs in LARP4 and -5/4b that contain Trp in place of the otherwise critical invariant Phe (8) found in all of the other ∼150 PAM2 sequences examined (1), which we refer to as PAM2w hereafter. We show that the LaM-RRM of human LARP4 preferentially binds poly(A) and exhibits other characteristics that suggest a recognition mode different from that of La proteins. Screening of a human cDNA library for yeast 2-hybrid interactions with LARP4 yielded RACK1, a 40S ribosome- and mRNA-associated kinase (17, 50), which was confirmed by reciprocal immunoprecipitations (IPs) from HeLa cells. LARP4 is cytoplasmic and interacts with PABP via two regions, PAM2w and a region following the RRM that includes ∼70 residues with significant homology to LARP5/4b. A peptide representing PAM2w of human LARP4 and the MLLE domain of PABP was examined by binding, nuclear magnetic resonance (NMR), and crystallography, which showed direct interactions similar to those of other PAM2-MLLE complexes. Further consistent with a translation-related function, LARP4 cosediments as two peaks on polysome profiles, with 40S ribosomes and with PABP on polysomes. After LARP4 knockdown, polysome profiles indicate deficiency in translation initiation, with [35S]methionine incorporation into newly synthesized protein diminished by ∼20%. LARP4 appears to promote translation, in part, by stabilizing mRNA, as suggested by our decay analyses.