A La protein requirement for efficient pre-tRNA folding

Abstract

The La protein protects the 3' ends of many nascent small RNAs from exonucleases. Here we report that La is required for efficient folding of certain pre-tRNAs. A mutation in pre-tRNA(Arg)(CCG) causes yeast cells to be cold-sensitive and to require the La protein Lhp1p for efficient growth. When the mutant cells are grown at low temperature, or when Lhp1p is depleted, mature tRNA(Arg)(CCG) is not efficiently aminoacylated. The mutation causes the anticodon stem of pre-tRNA(Arg)(CCG) to misfold into an alternative helix in vitro. Intragenic suppressor mutations that disrupt the misfolded helix or strengthen the correct helix alleviate the requirement for Lhp1p, providing evidence that the anticodon stem misfolds in vivo. Chemical and enzymatic footprinting experiments suggest a model in which Lhp1p stabilizes the correctly folded stem. Lhp1p is also required for efficient aminoacylation of two wild-type tRNAs when yeast are grown at low temperature. These experiments reveal that pre-tRNAs can require protein assistance for efficient folding in vivo.

Fig. 1. The trr4-1 mutation results in cold-sensitivity and a requirement for LHP1. (A) Positions of trr4-1 and trr4-2 mutations are shown on pre-tRNA^Arg_CCG. Leader and trailer lengths were estimated from pre-tRNA sizes on denaturing gels. (B) Five-fold serial dilutions of wild-type cells (TRR4 LHP1), cells lacking LHP1 (TRR4 lhp1Δ), trr4-1 cells lacking LHP1 (trr4-1 lhp1Δ) and trr4-1 cells carrying chromosomal (trr4-1 LHP1) or plasmid LHP1 (trr4-1 pLHP1) were spotted onto YPD agar and grown for four days at 25°C (top) or six days at 16°C (bottom). (C) RNA from wild-type cells (lane 1), cells lacking LHP1 (lane 2), trr4-1 cells with chromosomal or plasmid LHP1 (lanes 3 and 4) and trr4-2 cells with chromosomal or plasmid LHP1 (lanes 5 and 6) was subjected to northern analysis to detect tRNA^Arg_CCG (top). To detect pre-tRNAs, the blot was overexposed (middle). Asterisk, cross-hybridization with another isoacceptor. The blot was reprobed to detect tRNA^Ser_CGA (bottom). (D) A trr4-1 cell extract was incubated with pre-immune (lane 2) or anti-Lhp1p antibodies (lane 3). RNAs in the immunoprecipitate and an equivalent amount of extract (lane 1) were subjected to northern analysis to detect tRNA^Arg_CCG.

Fig. 2. Depletion of LHP1 from trr4 and trt2 cells. trt2-1 (A), trr4-1 (B) and trr4-2 cells (C) containing pMETLHP1 were grown at 25°C in media lacking methionine and switched to 2 mM methionine media at time 0. At intervals (lanes 3–8), RNA was subjected to northern analysis to detect tRNA^Thr_CGU (A, top; C, bottom), tRNA^Arg_CCG (A, bottom; B and C, top) or tRNA^Ser_CGA (B, bottom). RNA was also analyzed from wild-type (lanes 1) and lhp1Δ cells (lanes 2). For unknown reasons, tRNA^Thr_CGU runs as a doublet. (D) At intervals after the switch to 2 mM methionine, RNA was extracted from trr4-1 cells under acidic conditions and fractionated in acidic acrylamide gels. The northern blot was probed to detect tRNA^Arg_CCG and tRNA^Thr_CGU. Lane 1, deacylated trr4-1 RNA. RNA was also analyzed from wild-type cells (lane 2) and cells lacking LHP1 (lane 3). Consistent with an altered structure, charged and uncharged forms of wild-type tRNA^Arg_CCG migrate differently than these forms of trr4-1 tRNA^Arg_CCG. Lane 3 is underloaded.

Fig. 3. Aminoacylated tRNA^Arg_CCG declines when trr4-1 cells are grown at 16°C. (A) Wild-type (lanes 1–3) and trr4-1 cells carrying chromosomal (lanes 4–9) or plasmid LHP1 (lanes 10–16) were grown at 25°C and switched to 16°C at time 0. At indicated times, RNA was extracted and subjected to northern analysis to detect tRNA^Arg_CCG and tRNA^Thr_CGU. (B) At intervals, RNA was extracted under acidic conditions, fractionated in acidic acrylamide gels, and subjected to northern analysis to detect tRNA^Arg_CCG and tRNA^Thr_CGU.

Fig. 4. trr4-1 pre-tRNA adopts an altered conformation in vitro. (A) Wild-type (lanes 5–10) and trr4-1 pre-tRNA^Arg_CCG (lanes 11–16) (0.68 pmol each) were incubated with the indicated amounts of kethoxal in the absence or presence of 6.8 pmol Lhp1p. Modifications were detected by primer extension. Lanes 1–4, dideoxy sequencing of pre-tRNA^Arg_CCG. Lanes are labeled according to the RNA sequence. Each extension stop is one base below the modified nucleotide. In some experiments, modification of G26 is detected in wild-type pre-tRNA. (B) Wild-type (lanes 5–7) and trr4-1 pre-tRNA^Arg_CCG (lanes 8–10) were incubated with the indicated amounts of CMCT and subjected to primer extension. Lanes 1–4, dideoxy sequencing. (C) trr4-1 pre-tRNA^Arg_CCG was incubated in the absence (lanes 1–4) or presence (lanes 5 and 6) of Lhp1p. Proteinase K was added to digest Lhp1p (lanes 2, 4 and 6). Following a second incubation, kethoxal was added (lanes 3–6), and modifications detected as in (A). (D) The classic structure for pre-tRNA^Arg_CCG (left) and the proposed alternative structure (right). Sites of modification are designated by arrows (CMCT), triangles (kethoxal) and dots (DMS). Two G residues that are more accessible to kethoxal in wild-type RNA are red and two Gs that are less accessible are green. The alternative structure is predicted by MFOLD to be more stable than the classic structure by 2.3 kcal/mol. (E) 0.34 pmol of wild-type (lanes 1–6) and trr4-1 pre-tRNA^Arg_CCG (lanes 7–12) were incubated without protein (lanes 1–7) or with 2- (lanes 2 and 8), 4- (lanes 3 and 9), 6- (lanes 4 and 10), 8- (lanes 5 and 11) or 10-fold (lanes 6 and 12) molar excess of Lhp1p. RNA and RNPs were separated in native gels and detected by northern analysis. Asterisk, a second trr4-1 pre-tRNA conformer. (F) 0.68 pmol of trr4-1 (lanes 1–3) and trr4-1 pre-tRNA lacking the 3′ 9 nt (trr4-1,Δ3′) were incubated without protein (lanes 1 and 4), or with 5- (lanes 2 and 5) or 10-fold (lanes 3 and 6) molar excess of Lhp1p. Naked RNAs and RNPs were separated in native gels and detected by northern analysis. Asterisk, a conformer of trr4-1 pre-tRNA.

Fig. 5. Intragenic suppressor mutations favor formation of the correct helix. (A) Standard and proposed competing structures of pre-tRNA^Arg_CCG are shown along with positions of the trr4-1 (U31) mutation and A39 and U40 suppressors. (B) Top: 5-fold serial dilutions of wild-type, trr4-1 LHP1 and trr4-1 LHP1 strains carrying suppressors A39 (trr4-1,A39 LHP1) and U40 (trr4-1,U40 LHP1) were spotted on YPD agar and grown at 25°C for four days (left) or 16°C for six days (right). Bottom: growth of wild-type and trr4-1 strains lacking LHP1 at 25°C was compared with trr4-1 strains carrying mutations A39 (trr4-1,A39 lhp1Δ) and U40 (trr4-1,U40 lhp1Δ). (C) RNA from wild-type (lanes 1 and 5), trr4-1 (lanes 2 and 6), trr4-1,U40 (lanes 3 and 7) and trr4-1,A39 strains (lanes 4 and 8) containing LHP1 was subjected to northern analysis to detect tRNA^Arg_CCG (top) and tRNA^Thr_CGU (bottom). The band below mature tRNA^Arg_CCG in trr4-1,U40 strains may represent tRNA lacking CCA. (D) RNA was extracted under acidic conditions and fractionated in acidic acrylamide gels. The blot was probed to detect tRNA^Arg_CCG and tRNA^Thr_CGU. Charged species are indicated by dots. For trr4-1,U40, two species represent uncharged tRNA (lanes 4 and 8), one of which may lack CCA. Lane 1, deacylated TRR4 LHP1 RNA.

Fig. 6. LHP1 is required for efficient aminoacylation of wild-type tRNA^Arg_CCG. (A) Wild-type (lanes 5–12) and trr4-1 pre-tRNA^Arg_CCG (lanes 13–20) were incubated at the indicated temperature in the absence or presence of Lhp1p. Following incubation, 5 µl of kethoxal was added. Modifications were detected by primer extension. Lanes 1–4, dideoxy sequencing. The primer extension stops in the wild-type tRNA (lanes 5–12) at positions 30, 34 and 35 and near the top of the gel are not kethoxal-dependent. (B) Wild-type (lanes 1–7) and cells lacking LHP1 (lanes 8–12) were grown at 25°C and switched to 16°C at time 0. At intervals, RNA was extracted under acidic conditions and subjected to northern analysis to detect tRNA^Arg_CCG (top), tRNA^Thr_CGU (middle) and tRNA^Gln_CUG (bottom panel). Lane 1, deacylated wild-type RNA.

Fig. 7. Lhp1p may contact the acceptor stem and anticodon loop of pre-tRNA^Arg_CCG. (A) 5′ labeled pre-tRNA^Arg_CCG was incubated without (lanes 3, 5, 7 and 9) or with (lanes 4, 6, 8 and 10) Lhp1p, followed by cleavage with T1 (lanes 5 and 6), T2 (lanes 7 and 8) or V1 (lanes 9 and 10) ribonucleases. In the experiment, ∼85% of the labeled RNA was bound by Lhp1p. Lanes 1 and 2, T1 ribonuclease and alkaline hydrolysis ladders. Asterisks, sites of weak protection by Lhp1p. (B) Phosphorimager quantitation of V1 cleavage of the acceptor stem (top) and the anticodon stem (bottom). (C) Quantitation of T2 cleavage of the anticodon loop (top) and D loop (bottom). (D) Pre-tRNA^Arg_CCG was modeled on the structure of tRNA^Arg_ICG (Delagoutte et al., 2000) using SPOCK. Bases protected from cleavage by Lhp1p are shown in red.