Precursor-product discrimination by La protein during tRNA metabolism

Abstract

La proteins bind pre-tRNAs at their UUU-3'OH ends, facilitating their maturation. Although the mechanism by which La binds pre-tRNA 3' trailers is known, the function of the RNA binding beta-sheet surface of the RNA-recognition motif (RRM1) is unknown. How La dissociates from UUU-3'OH-containing trailers after 3' processing is also unknown. Here we show that La preferentially binds pre-tRNAs over processed tRNAs or 3' trailer products through coupled use of two sites: one on the La motif and another on the RRM1 beta-surface that binds elsewhere on tRNA. Two sites provide stable pre-tRNA binding, whereas the processed tRNA and 3' trailer are released from their single sites relatively fast. RRM1 loop-3 mutations decrease affinity for pre-tRNA and tRNA, but not for the UUU-3'OH trailer, and impair tRNA maturation in vivo. We propose that RRM1 functions in activities that are more complex than UUU-3'OH binding. Accordingly, the RRM1 mutations also impair an RNA chaperone activity of La. The results suggest how La distinguishes precursor from product RNAs, allowing it to recycle onto a new pre-tRNA.

Figure 1

La can bind non-UUU-3OH-containing RNA via contacts that are not mediated by the previously characterized RNA 3'OH binding site in the La motif. (a) EMSAs reveal human La protein (hLa) binding to the 12 nt UUU-3’OH-containing trailer and (b) a tRNA^ArgACG transcript which lacks UUU-3’OH. (c & d) show binding by the mutated protein hLa-Q20A/Y23A/D33R (abbreviated hLa-QYD) that contains three substitutions in the LM that are known to be critical for UUU-OH-specific binding (see text). For each, a constant trace amount of ³²P-RNA (~0.1 nM) was incubated with varying concentrations of La protein as indicated above the lanes in nM.

Figure 2

Two distinct RNA binding sites on La together enhance stable binding to pre-tRNA. (a–c) Scatchard analyses of Method 2 EMSAs performed on pre-tRNA^ArgACG, tRNA^ArgACG and the 12 nt UUU-3'OH trailer at 0, 10 and 1 mM Mg²⁺. Each titration was done at a constant concentration of La with concentrations of RNA varying, although the La concentration differed for each individual Scatchard plot; K_ds are provided in each panel next to each ligand and in panel G. (d–f) Analysis of dissociation of hLa from pre-tRNA^ArgACG, tRNA^ArgACG and the 12 nt UUU-3'OH trailer from data derived from EMSAs, was performed and analyzed as described. Time scales are 0 to 350 seconds. (g) K_ds derived from A–C above and the numerical fraction of the K_ds at 0 and 10 mM Mg²⁺ (0 Mg/10 Mg) as indicated. Standard errors derived from triplicate determinations for pre-tRNA^ArgACG, tRNA^ArgACG and the 12 nt UUU-3'OH trailer were 14.7%, 11.7% and 10.9%, respectively in 0 Mg²⁺, 5.5%, 7.3% and 8.9%, respectively in 10 mM Mg²⁺, and 7.7%, 5.9% and 13.9% in 1 mM Mg²⁺. k_offs were derived from D–F above using standard calculations. The t_1/2s were then derived from k_off. Standard errors for dissociation of pre-tRNA^ArgACG, tRNA^ArgACG and the 12 nt UUU-3'OH trailer were 8.5%, 7.1% and 10.8%, respectively. The units for k_off and t_1/2 are min⁻¹ and min, respectively.

Figure 3

hLa exhibits strong preference for pre-tRNA over a UUU-3'OH trailer. (a & b) Binding reactions contained constant amounts of hLa protein (100 nM) and a trace amount of ³²P-RNA (~0.1 nM, pre-tRNA in A, or UUU-3'OH trailer in (a), and varying amounts of unlabelled (cold) competitor RNAs as indicated above the lanes and described in the text, in 1 mM Mg²⁺. The data in A and B were quantified and analyzed in (c) and (d) respectively.

Figure 4

La RRM1 loop-3 mediates UUU-3’OH-independent tRNA binding. (a) Sequence alignment of the β2-loop 3-β3 regions of the RRM1s of the La proteins from seven organisms followed by the homologous regions of U1A and PABP. Asterisks indicate the basic residues conserved in loop 3 of La proteins. RNP1 is indicated; in hLa this contains F155, indicated by vertical line above the sequence. (b) Structure of the loop-3 side chains relative to the RRM1 β-sheet surface (adopted from Kotik-Kogan et al., PDB accession # 2VON). The loop-3 basic residues mutated for this study are shown in blue. RRM1 is shown in green, with the conserved aromatic residue side chains (Y114 and F155) on β-strands 1 and 3, in cyan.

Figure 5

The hLa loop mutant is defective in tRNA maturation in vivo. (a) tRNA-mediated suppression (TMS) activity was assayed in S. pombe; pRep is the empty vector; all other hLa constructs indicated were cloned in pRep,,,. (b) Northern blot analysis of RNAs extracted from cells in A above. Upper panel shows blot probed for intron-containing pre-tRNA^LysCUU species, which migrate as upper, middle and lower bands (see text). Lower panel shows the same blot reprobed for U5 snRNA, which served as a loading control for quantitation, normalized to hLa = 1.0, lane 3. (c) RNA chaperone assays were performed in vitro using the cis-splicing intron RNA. Activity is reflected by a decrease in unspliced RNA (ln U/Uo, Experimental Procedures) for each of the proteins indicated. Error bars reflect triplicate samples for each point. (d) Coomassie blue stained gel of the BSA (lane 1), hLa (lane 2) and hLa-loop (lane 3) proteins used in the assay.

Figure 6

Model of involvement of La protein in a tRNA maturation pathway. La appears to be the first protein that binds nascent pol III-transcripts including pre-tRNAs (see text), and presumably does so via its UUU-3'OH binding cleft and/or RRM1. Different regions of the RNA may become juxtaposed to RRM1 as the pre-tRNA folds, acquires nucleotide modifications (indicated by asterisks), and becomes a substrate for 5' processing by RNase P. After separation of the tRNA and 3' trailer by cleavage by the endonuclease, RNase Z, La is no longer tethered to two sites on a single RNA and readily dissociates, free to associate with new nascent pre-tRNA. The released, end-matured, modified tRNA becomes the substrate of 3' end-modifying proteins such as the CCA-adding enzyme and tRNA synthetases, and is exported from the nucleus.