Functional analysis of human tRNA isodecoders

Abstract

tRNA isodecoders share the same anticodon but have differences in their body sequence. An unexpected result from genome sequencing projects is the identification of a large number of tRNA isodecoder genes in mammalian genomes. In the reference human genome, more than 270 isodecoder genes are present among the approximately 450 tRNA genes distributed among 49 isoacceptor families. Whether sequence diversity among isodecoder tRNA genes reflects functional variability is an open question. To address this, we developed a method to quantify the efficiency of tRNA isodecoders in stop-codon suppression in human cell lines. First, a green fluorescent protein (GFP) gene that contains a single UAG stop codon at two distinct locations is introduced. GFP is only produced when a tRNA suppressor containing CUA anticodon is co-transfected with the GFP gene. The suppression efficiency is examined for 31 tRNA isodecoders (all contain CUA anticodon), 21 derived from four isoacceptor families of tRNASer genes, 7 from five families of tRNALeu genes, and 3 from three families of tRNAAla genes. We found that isodecoder tRNAs display a large difference in their suppression efficiency. Among those with above background suppression activity, differences of up to 20-fold were observed. We were able to tune tRNA suppression efficiency by subtly adjusting the tRNA sequence and inter-convert poor suppressors into potent ones. We also demonstrate that isodecoder tRNAs with varying suppression efficiencies have similar stability and exhibit similar levels of aminoacylation in vivo. Our results indicate that naturally occurring tRNA isodecoders can have large functional variations and suggest that some tRNA isodecoders may perform a function distinct from translation.

Figure 1. The experimental set-up and dosage optimization

(A) Schematic description of the method used for the evaluation of the different tRNA scaffolds. (B) Fluorescence recovery versus the amount of suppressor tRNA^Ser transfected. Identical amount of reporter plasmid encoding GFP S29stop is used in all transfections. Fluorescent values reported here are obtained from the average of 4 independent transfections. The dashed line indicates the observed fluorescence when equivalent amount of plasmid encoding wild type GFP is used.

Figure 2. Suppression analysis of isodecoders derived from isoacceptor families of leucine, serine and alanine

(A) Detailed organization of tRNA isoacceptor families. Number of genes and isodecoders in each isoacceptor family is indicated. (B) Structural alignment of representative isodecoders of each isoacceptor family. Representatives correspond to tRNA encoded by the highest number of gene copies. Light grey box indicates position of the anticodon. Dark grey box highlights position 32 and 67 where mutations have been made. (C) Suppression efficiency for the 12 Leu, Ser and Ala-tRNA specific scaffolds tested. 100% corresponds to the most active scaffold (Leu(CAA)). Efficiency is proportional to fluorescence recovery. GFP S29stop is used as a reporter.

Figure 3. Sequence swap between two tRNA^Leu suppressors

(A) Cloverleaf representation (CAG and CAA scaffolds). Common nucleotides are in light grey. Mutations are indicated by the arrows. (B) Suppression efficiency of the four corresponding suppressors. Backbone and nucleotides 32 and 67 are indicated below.

Figure 4. Sequence alignment of isodecoders from all tRNA^Ser genes

(A) Detailed organization of the complete set of tRNA^Ser species in the reference human genome. The number of gene copies and the sequence of the original anticodon are indicated. (B) Sequence alignment, ordered according to their suppression efficiency (data in figure 5). Anticodon triplet is boxed. Sequence S19 is not aligned due to its non-canonical folding.

Figure 5. Suppression analysis of isodecoders derived from tRNA^Ser genes

(A) Suppression efficiency for the 19 tRNA^Ser specific scaffolds tested. 100% corresponds to the most active scaffold (S01). GFP S65stop is used as a reporter for simultaneous evaluation of both charging specificity and ranking of suppression efficiency. S01, S07 and S15 were chosen as representatives of high, medium and low suppressors respectively and used for all further studies. The beads on strings indicate the sequence of the original anticodon before substitution by suppressor CUA triplet. (B) Suppression efficiency for previously tested S01, S07 and S15 suppressors. GFP S29stop is used as a reporter. Position 29 can accommodate any amino acid without disturbing light emission. (C) Cloverleaf diagram of S01, S07 and S15. Sequence changes between S01 (high efficiency suppressor) and the mid (S07) and low (S15) efficiency suppressor are in black (common nucleotides appear in grey).

Figure 6. Comparing in vivo stability of high, medium and low efficiency suppressors

(A) S01, S07 and S15 scaffolds show equivalent stability when incubated in human cells. The 3′ terminal A of corresponding tRNAs has been ³²P-labeled using CCA-adding enzyme and lipofected in HeLa cells. Total RNAs have been extracted at different time points and separated on denaturating PAGE. (B) Fraction of radioactive signals for the tRNA band at different time points shows similar half-lives for these suppressor-tRNAs. It corresponds to the ratio of the radioactive signal specific to full-length tRNA over the signal of the entire lane.

Figure 7. Comparing in vivo charging levels of high, medium and low efficiency suppressors

³²P-labeled S01, S07 and S15 scaffolds (label at 3′A) are incubated in HeLa cells, extracted under acidic conditions, digested by nuclease P1 and separated by TLC. Spots corresponding to free and acylated [³²P]-pA are indicated. Right panel shows control with Wild Type (WT) transcripts of tRNA^Ser (AGA) treated the same way. Suppressors and native tRNA^Ser show comparable aminoacylation levels in vivo.

Figure 8. Comparing folding in solution of high, medium and low efficiency suppressors

Structural mapping of in vitro transcribed wild-type tRNA^Ser(AGA) and suppressors S01, S07 and S15 by limited nuclease S1 and V1 digestion. Cleavage products of 5′-labeled transcripts are separated by electrophoresis on a 10% polyacrylamide-8M urea gel and visualized by phosphorimaging. The two first lines correspond to alkaline (BH) and RNase T1 hydrolysis ladder of transcripts S01. Transcripts and the nature of treatment are indicated on top of each line. Some tRNA structural units are indicated on the side of the gel.

Figure 9. Expansion of the loop size in the variable arm significantly increases suppression efficiency

(A) Nucleotides have been grafted in the loop of the hairpin in the variable arm of S07 scaffold. (B) Suppression efficiency of wild type and two grafting mutants are indicated. GFP S65stop is used as a reporter. (C) In vivo charging analysis of these three transcripts shows comparable aminoacylation levels.