Human La binds mRNAs through contacts to the poly(A) tail

Abstract

In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3'OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail.

Figure 1.

Human La displays length dependent affinity for poly(A). (A) Binding of recombinant human La for U10, U20, A10 and A20 was tested by electromobility shift assay (EMSA) using HepC hairpin RNA as competitor; gels for C10, C20, G10 and G20 provided in Supplementary Figure S1. (B) Graphical representation of EMSA results. (C) EMSA of human La for U10 and A20 in the presence of C10 as competitor. (D) Graphical representation of binding curves comparing La affinity for U10 and A20 with C10 versus HepC hairpin competitors.

Figure 2.

Uridylate containing RNAs compete poorly for poly(A) binding to hLa. Sufficient recombinant human La was added (2 μM) to radiolabelled A20 to achieve >95% binding (NR: no cold RNA added) and increasing concentrations of various cold RNAs were added to assess their ability to displace A20 from hLa. (A) Comparison of U10 (left-hand series) and A20 (right-hand series) for radiolabelled A20 binding. (B) Comparison of A10 (left-hand series) and U20 (right-hand series) for radiolabelled U20 binding. NP: no recombinant hLa protein added (i.e. free radiolabeled A20 or U20). ‘1’: indicative of 1:1 La–RNA complex; ‘2’: indicative of multimeric La–RNA complex. * (asterisk): new RNP formed between La and A20 with addition of U10.

Figure 3.

The winged-helix face of the La motif is involved in adenylate binding. (A) Structure of La motif (11) with uridylate binding amino acids labeled in red, UUU-3’OH RNA in cyan, winged helix face of La motif in yellow and mutated amino acids of winged-helix face in green. (B) EMSAs of hLa Q20A/Y23A/D33R binding to U10 and A20. (C) Graphical representation of hLa and hLa Q20A/Y23A/D33R bound to U10 and A20. (D) EMSAs of hLa K86A/T87A/K88A binding to U10 and A20. (E) Graphical representation of hLa and hLa K86A/T87A/K88A bound to U10 and A20.

Figure 4.

Human La binds to poly(A) in human cells. (A) Crosslinking immunoprecipitation of transfected myc-PABP and GFP-hLa or GFP-hLa mutants in HEK293T cells. Left: limiting digestion with RNase I; right: Digestion with RNase T1. IP: immunoprecipitation. Presence of poly(A) or pre-tRNA Met-e in immunoprecipitated RNPs was assessed by Northern blot. Bottom: Western blot confirming transfection of myc-PABP or GFP-hLa. (B) Co-immunoprecipitation of cyclin D (CCND1) mRNA, BiP mRNA or 5.8S rRNA with GFP-hLa 1–375 versus GFP-hLa 1-375 K86A/T87A/K88A relative to GFP vector only control as measured by qPCR. Error bars correspond to standard error of the mean, asterisks highlight statistically significant changes (*P-value < 0.05; ***P-value < 0.001 two-tailed Student's t-test).

Figure 5.

The poly(A) binding mode promotes hLa entry into polysomes. Top: Trace of ribosomes/polysomes fractionated from HEK293T cells transfected with indicated constructs or treated with puromycin. Bottom: Western blots versus Rpl9 and GFP-hLa, GFP hLa 1–375 and indicated mutants, as well as a puromycin treated control for GFP hLa 1–375.

Figure 6.

The human La poly(A) binding mode contributes to La function in translation. (A) Left: Schematic of bicistronic reporter construct used for direct transfection into HEK293T cells. Right: Western blots of transfected myc-PABP, GFP-hLa, GFP-hLa 1–375 or K86A/T87A/K88A mutants of these. GAPDH shown as loading control. (B) Relative expression of cap-dependent (left; renilla) and cap-independent (right; firefly) reporter genes in the presence of overexpressed GFP-hLa, GFP-hLa 1–375 or the K86A/T87A/K88A mutants on the 20A or 40A tailed mRNA reporter constructs normalized to level expression in GFP vector control. (C) Enhanced expression from bicistronic reporter mRNAs upon co-expression of GFP-hLa 1–375 is not due to enhanced reporter mRNA stability. Total RNA was isolated and levels of transfected 20A or 40A bicistronic reporters were assessed by qPCR 8 h post-transfection in cells that had been previously transfected by indicated GFP or GFP-hLa 1–375 constructs. Reporter mRNA levels are provided relative to amounts in the GFP-vector transfected cells after normalization for total RNA abundance via qPCR for the U5 snRNA. (D) Effect of overexpression of myc-PABP on the GFP-hLa and GFP-hLa 1–375 associated expression of cap-dependent (left, renilla) and cap-independent (right, firefly), normalized to the expression levels in the context of the GFP vector control ± overexpression of myc-PABP. (E) Ratios of renilla/luciferase expression in the context of GFP-hLa or GFP-hLa 1–375 expression ± the expression of myc-PABP. (*P-value < 0.05; ** P-value <0.01; ***P-value < 0.001). Error bars: SEM.