Queuosine is incorporated into precursor tRNA before splicing

Abstract

Each newly transcribed tRNA molecule must undergo processing and receive modifications to become functional. Queuosine (Q) is a tRNA modification present at position 34 of four tRNAs with "GUN" anticodons. Among these, the precursor of tRNA^Tyr carries an intronic sequence within the anticodon loop that is removed by an essential non-canonical splicing event. The functional and temporal coupling between tRNA-splicing and Q-incorporation remains elusive. Here, we demonstrate in vitro and in vivo that intron-containing precursors of tRNA^Tyr are modified with Q or with the Q-derivative galactosyl-queuosine (galQ) before being spliced. We show that this order of events is conserved in mouse, human, flies and worms. Using single particle cryo-EM, we confirm that pre-tRNA^Tyr is a bona fide substrate of the QTRT1/2 complex, which catalyzes the incorporation of Q into the tRNA. Our results elucidate the hierarchical interplay that coordinates Q-incorporation and splicing in eukaryotic tRNAs, providing a relevant but unappreciated aspect of the cellular tRNA maturation process.

Fig. 1. tRNA^Tyr is queuosinylated at precursor level in mESCs and tissues.

a G₃₄ of tRNA^Tyr is modified with queuosine (Q) and galactosyl-queuosine (galQ) by the sequential action of the QTRT1/2 complex (TGT) and QTGAL enzyme. Red rectangle indicates the cis-diol group reacting in APB Northern blot. b Schematic representation of the probes used in the Northern blots mapped onto the standard cloverleaf structure of tRNAs. The empty circles represent G₃₄, the red triangles indicate the splicing sites. Pre-tRNA probes are designed complementary to the intron and detect only precursor tRNAs containing introns. tRNA probes are designed complementary to the 5’end of specific tRNAs and can detect both pre- and mature tRNAs. c GalQ on mature tRNA^Tyr cannot be resolved in APB Northern blot, which shows only residual Q modification in mESCs. d Expression of pre-tRNA^Tyr is quantified by Northern blot (Supplementary Fig. 1b) in mESCs and in the mouse brain. e APB Northern blot showing queuosinylation of tRNA^Tyr precursors in wild type mESCs with a pool of probes targeting pre-tRNA^Tyr 1-5, 2-1, 3-2. Qtrt1(Q1) and Qtrt2 (Q2) knockout cell lines are shown as negative control. f Specific probes against mouse pre-tRNA^Tyr 1-4 (left panel) and pre-tRNA^Tyr 2-1 (middle panel) show that these specific precursors are modified already at precursor level in wild type mESC. g Specific probes against mouse tRNA^Tyr precursors show that pre-tRNA^Tyr 1-4 is modified with Q already before the splicing in mouse brain; galQ on mature tRNA^Tyr cannot be resolved in APB northern blot (left panel). Red arrows indicate Q, black arrows indicate unmodified or unresolved galQ, wt: wild type, Q1: Qtrt1^−/−, Q2: Qtrt2^−/−.

Fig. 2. mQTRT1/2 complex recognizes and modifies in-vitro transcribed and native pre-tRNA^Tyr.

a Schematic representation of in-vitro transcribed RNA according to the canonical cloverleaf structure of pre-tRNA and tRNA used in combination of mQTRT1/2 complex to assess the enzymatic specificity. The circles represent position 34, the triangles indicate the splicing sites. IM: intron mutant. AM: anticodon mutant. I: intron replacement. b, c The activity of the mQTRT1/2 complex is evaluated by LC-MS/MS on the same amount of injected RNA, measuring the incorporated queuine using the indicated in-vitro pre- and mature tRNAs after in vitro Q modification. tRNA^Leu/pre-tRNA^Leu and tRNA^Tyr are used as negative and positive control, respectively. mQTRT1/2 complex 200 nM, queuine 5 µM, 3 h at 37 °C. d The activity of mQTRT1/2 complex on bulk tRNA isolated from Q1 or Q2 mESCs is evaluated by LC-MS/MS, measuring the incorporated queuine after in vitro Q assay. Q1, Q2 and wt tRNAs are used as negative and positive control, respectively. e APB Northern blot of the samples indicated in d showing the activity of recombinant mQTRT1/2 complex (mTGT) on native pre-tRNA^Tyr 1-4. Red arrows indicate Q, black arrows indicate unmodified or unresolved galQ, wt: wild type, Q1: Qtrt1^−/−, Q2: Qtrt2^−/−.

Fig. 3. Cryo-EM structures of mQTRT1/2 with tRNA^Tyr and pre-tRNA^Tyr 1-4.

a Representative SDS-PAGE analysis of purified human and mouse QTRT1/2 complexes. MW: molecular weight marker. b MST analyses of mQTRT1/2 binding to tRNA^Tyr and pre-tRNA^Tyr 1-4. Concentration of mQTRT1/2 and calculated K_d values (K_d values) are given, concentration of tRNA is kept constant at 50 nM. Individual measurements at each concentration are shown with the fitted curve. Error bars represent ±SD (n = 3, biological replicates). c EMSA analyses of mQTRT1/2 binding to Cy5-labeled tRNA^Tyr and pre-tRNA^Tyr 1-4. Protein concentrations are given, concentration of tRNA is kept constant at 200 nM. d Overview of structures of mQTRT1/2 complexes with tRNA^Tyr (left) or pre-tRNA^Tyr 1-4 (right). Representative 2D class averages (top) and cryo-EM maps are shown next to the respective atomic models (bottom). e Close-up view on the anticodon stem loop (ASL) region bound to the QTRT1 active site in mQTRT1/2 with tRNA^Tyr (left), mQTRT1/2 with pre-tRNA^Tyr 1-4 (middle) and hQTRT1/2 with tRNA^Asp (PDB ID 8OMR; right). Anticodon loop G₃₄ (AP-34) is in green, 9-deazaguanine (9dG) is visible in the active sites.

Fig. 4. pre-tRNA^Tyr 1-2 is modified at G₃₄ with galQ before splicing.

a The presence of galactose modification on Q₃₄ can eliminate its capacity to interact with boronic acid derivative in the ABP-PAGE. β1-3,4 Galactosidase (β-GAL) is used to hydrolyze terminal β-galactose residues from the precursor and mature tRNA^Tyr. Blue circles indicate the secondary amine protonated and reacting in AD Northern blot. Red rectangle indicates the cis-diol group reacting in APB Northern. b Both pre-tRNA^Tyr1-4 and 1-2 are shifted in wild type mESCs using AD Northern indicating the presence of a Q derivative modification. c Digestion of galQ using β1-3,4 Galactosidase (β-GAL) revealed the Q derivative modification on mature m_tRNA^Tyr and m_pre-tRNA^Tyr1-2 in mESCs and the mouse brain using gdAPB Northern blot.

Fig. 5. Evolutionary analysis of the pre-tRNA^Tyr modification at position 34 using Northern blot.

a galQ modification is detected on mature h_tRNA^Tyr and on h_pre-tRNA^Tyr 8-1 and 9-1 in human HeLa cells grown in medium ± q using gdAPB Northern blot. b D. melanogaster is missing QTGAL enzyme and Q modification is detected on d_pre-tRNA^Tyr 1-4 in S2 cells grown in medium ± q using APB Northern blot. c Q modification is detected on mature c_tRNA^Tyr and on c_pre-tRNA^Tyr 2-8 in N2 wild type strain of C. elegans grown in peptone rich medium (LB) versus NGM plates (N) using gdAPB Northern blot. Red arrows indicate Q, black arrows indicate unmodified or unresolved galQ.