Independent suppression of ribosomal +1 frameshifts by different tRNA anticodon loop modifications

Abstract

Recently, a role for the anticodon wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) has been revealed in the suppression of translational +1 frameshifts in Saccharomyces cerevisiae. Loss of either the mcm⁵U or s²U parts of the modification elevated +1 frameshift rates and results obtained with reporters involving a tRNA^Lys_UUU dependent frameshift site suggested these effects are caused by reduced ribosomal A-site binding of the hypomodified tRNA. Combined loss of mcm⁵U and s²U leads to increased ribosome pausing at tRNA^Lys_UUU dependent codons and synergistic growth defects but effects on +1 frameshift rates remained undefined to this end. We show in here that simultaneous removal of mcm⁵U and s²U results in synergistically increased +1 frameshift rates that are suppressible by extra copies of tRNA^Lys_UUU. Thus, two distinct chemical modifications of the same wobble base independently contribute to reading frame maintenance, loss of which may cause or contribute to observed growth defects. Since the thiolation pathway is sensitive to moderately elevated temperatures in yeast, we observe a heat-induced increase of +1 frameshift rates in wild type cells that depends on the sulfur transfer protein Urm1. Furthermore, we find that temperature-induced frameshifting is kept in check by the dehydration of N6-threonylcarbamoyladenosine (t⁶A) to its cyclic derivative (ct⁶A) at the anticodon adjacent position 37. Since loss of ct⁶A in elp3 or urm1 mutant cells is detrimental for temperature stress resistance we assume that conversion of t⁶A to ct⁶A serves to limit deleterious effects on translational fidelity caused by hypomodified states of wobble uridine bases.

Keywords: 5-methoxycarbonylmethyl-2-thiouridine; cyclic N6-threonylcarbamoyladenosine; tRNA modification; translation; translational frameshift.

Figure 1.

Anticodon loop modifications of tRNA^Lys_UUU and translational +1 frameshift models. (A) Schematic representation of tRNA^Lys_UUU anticodon loop with ct⁶A (6) and mcm⁵s²U (*) modifications. Gene-products relevant for synthesis of the modifications and addressed in this study are indicated. (B) Schematic representation of the modified Ty1 frameshift construct sensitive to tRNA^Lys_UUU modification defects. Shown is the W12 construct where lacZ is in the +1 frame with respect to HIS4A. The shift site as well as amino acids specified and +1 and 0 frame UGA stop codons are indicated. (C) With fully modified tRNA^Lys_UUU frameshift rates are low and termination at the UGA downstream of the Lys codon occurs efficiently. (D) Frameshift induction by tRNA^Lys_UUU modification defects via an A-site effect. Hypomodified tRNA^Lys_UUU (absence of * and 6, stippled) inefficiently binds to the AAA codon while peptidyl-tRNA^Leu_UAG occupies the P-site. This pausing allows the peptidy-tRNA^Leu_UAG to shift to the +1 frame and the appearance of an AAC codon in the A site (read by tRNA^Asn_GUU). This effect would be suppressible by extra copy of tRNA^Lys_UUU. (E) Frameshift induction by tRNA^Lys_UUU modification defects via a P-site effect. Hyopmodified tRNA^Lys_UUU (absence of * and 6) binds to the AAA codon and translocation occurs. Once present in the P-site as peptidyl tRNA^Lys_UUU, binding is weakened (loosing P-site grip) and +1 frameshift occurs. This effect would not be suppressible by extra copy of tRNA^Lys_UUU.

Figure 2.

Induction of +1 translational frameshift rates relative to the wild type. Fold induction rates were calculated based on normalized frameshift values [%] shown in Table 1 and significance of differences determined using two-tail t-test. n.s.: not significantly different. Heat indicates moderate thermal stress condition during growth (37°C) and + tK(UUU) refers to frameshift levels measured with the WA-2 and FA-1 constructs that carry an additional copy of the tK(UUU) gene.

Figure 3.

Effect of temperature shift on Ahp1 urmylation and Urm1 levels. (A) Sulfur transfer from cysteine to Urm1 results in formation of Urm1 thiocarboxylate, which is required for covalent attachment of Urm1 to Ahp1 (urmylation) and tRNA thiolation. (B) Temperature shift assay. urm1 mutants expressing TAP-tagged Urm1 were cultivated at 30°C in YNB media to OD_600nm = 1 after which one half of the culture was shifted to 37°C. At indicated time points, total protein extracts were prepared and subjected to anti-TAP Western analysis to detect free Urm1 as well as the slower migrating Ahp1-Urm1 conjugate. Previous studies with ahp1mutants confirmed the the slower migrating band to result from Ahp1 urmylation by the tagged Urm1 variant. To verify equal loading, anti-Cdc19 antibodies were utilized. (C) As in (B) but with the culture part that remained at 30°C. (D) Signal intensities of slower migrating band in (B) and (C) were quantified and relative levels of Ahp1 urmylation at 30°C or 37°C were calculated by dividing signal intensity at time points 2.5h, 5h or 7.5h by the one of timepoint 0.

Figure 4.

Heat induced growth defects in tcd1 and tcd2 single mutants are suppressible by elevated levels of tRNA^Lys_UUU. tcd1 and tcd2 mutants were transformed with either pRS425 (vector), pK (multi copy tRNA^Lys_UUU) or pQKE (multi copy tRNA^Lys_UUU, tRNA^Gln_UUG and tRNA^Glu_UUC) and serial dilutions of cultures transferred to YPD plates that were incubated at the indicated temperatures for two days.