Examining tRNA 3'-ends in Escherichia coli: teamwork between CCA-adding enzyme, RNase T, and RNase R

Abstract

tRNA maturation and quality control are crucial for proper functioning of these transcripts in translation. In several organisms, defective tRNAs were shown to be tagged by poly(A) or CCACCA tails and subsequently degraded by 3'-exonucleases. In a deep-sequencing analysis of tRNA 3'-ends, we detected the CCACCA tag also in Escherichia coli However, this tag closely resembles several 3'-trailers of tRNA precursors targeted for maturation and not for degradation. Here, we investigate the ability of two important exonucleases, RNase R and RNase T, to distinguish tRNA precursors with a native 3'-trailer from tRNAs with a CCACCA tag. Our results show that the degrading enzyme RNase R breaks down both tRNAs primed for degradation as well as precursor transcripts, indicating that it is a rather nonspecific RNase. RNase T, a main processing exonuclease involved in trimming of 3'-trailers, is very inefficient in converting the CCACCA-tagged tRNA into a mature transcript. Hence, while both RNases compete for trailer-containing tRNA precursors, the inability of RNase T to process CCACCA tails ensures that defective tRNAs cannot reenter the functional tRNA pool, representing a safeguard to avoid detrimental effects of tRNAs with erroneous integrity on protein synthesis. Furthermore, these data indicate that the RNase T-mediated end turnover of the CCA sequence represents a means to deliver a tRNA to a repeated quality control performed by the CCA-adding enzyme. Hence, originally described as a futile side reaction, the tRNA end turnover seems to fulfill an important function in the maintenance of the tRNA pool in the cell.

Keywords: RNase R; RNase T; tRNA decay; tRNA maturation; tRNA quality control.

FIGURE 1.

Abundance and recognition of tRNA CCACCA extensions as well as 3′-trailer sequences in E. coli. (A) Boxplots of the fraction of E. coli CA244 wt and Δcca tRNAs during exponential (log) and stationary (stat) growth carrying CCACCA extensions. Values are given as a fraction of respective tRNA subpopulations normalized to the total tRNA reads for each isoacceptor. (B) Nucleotide frequencies within the 3′-trailer region of the E. coli K12 tRNA genes starting from the CCA triplet (weblogo.berkeley.edu). Letter height correlates with abundance; A and U residues are highlighted in gray. (C) Radiolabeled E. coli tRNA^Ala with either its native 3′-trailer (CCAAAU, left) or the corresponding degradation tag (CCACCA, right) was incubated with either RNase R (upper panel, 20 ng µL⁻¹) or RNase T (lower panel, 0.6 ng µL⁻¹). Assays were carried out in a 40 µL total reaction volume with 5 µM tRNA substrate. C, control reactions without enzyme incubated for the maximal reaction time; M, size marker tRNA^Ala, 73 nt.

FIGURE 2.

RNase R activity on tRNA substrates with 3′-overhangs of similar length. (A) Time-dependent degradation of internally labeled tRNA^Ala–CCACCA and tRNA^Ala–CCAAAU yield almost identical curves, implying that RNase R does not discriminate between the two 3′-overhangs; n = 3. (B) Competition studies with tRNA^Ala–CCACCA and tRNA^Ala–CCAAAU of the RNase R catalyzed reaction show identical inhibition curves, indicating that the enzyme accepts both substrate overhangs independent of their sequence composition. No comp., control reaction in the absence of competitor. Data are means ± SD; n = 3.

FIGURE 3.

RNase T kinetics on tRNA substrates with 3′-overhangs of similar length. (A) Our initial experiments showed that tRNAs are degraded to different extents, depending on the sequence of the 3′-extension (reaction velocity is indicated by size and color of the individual arrows; experimental data are not shown). For the kinetic analysis, main reaction products able to reenter the functional tRNA pool were taken into account (3′-end in bold characters). tRNA–CCAAAU with the natural trailer (left) is rapidly degraded to the CCA end as the main reaction product (green arrow) that was analyzed in the kinetic analysis. The subsequent 3′-end turnover (removal of the terminal A residue, orange arrow) is slower and therefore was not considered. Individual bases of the degradation tag CCACCA are removed at different reaction velocities (right). Although the terminal A residue of the CCACCA sequence is also removed very fast (green arrow), degradation of the two following C residues is very slow (red arrow) and corresponds to the rate-limiting step of the reaction. The subsequent 3′-end turnover (removal of the terminal A residue of the resulting CCA end, orange arrow) is then removed very efficiently. Hence, for this tRNA, tRNA–CC represents the resulting main reaction product for the kinetic analysis. (B) Kinetic analysis of tRNA^Ala–CCAAAU trailer removal by RNase T. The CCACCA tag is nearly not converted. k is the reaction rate normalized to the total enzyme concentration. Data are means ± SD; n = 3–4. A different scale of the kinetics of the CCACCA removal is displayed in Supplemental Figure S2.

FIGURE 4.

RNase T-catalyzed tRNA end turnover. (A) Time-dependent 3′-end turnover of a 5′-end labeled tRNA^Ala transcript ending with the CCA triplet. RNase T specifically catalyzes the removal of the terminal A residues and leaves the CC sequence intact. C, control reactions incubated without enzyme for the maximum reaction time; M, size marker tRNA^Ala, 73 nt. (B) Apparent kinetics of tRNA 3′-end turnover. Increasing amounts of tRNA^Ala–CCA were incubated with RNase T under steady-state conditions and product formation was determined by quantifying the intensity of the tRNA^Ala–CC band. Michaelis–Menten parameters were determined using GraphPadPrism software (Table 2); k is the reaction rate normalized to the total enzyme concentration. Data are means ± SD; n = 3.

FIGURE 5.

Interlocked quality control and processing of tRNAs in E. coli. After transcription, a set of endo- and exonucleases remove the 5′-leader and 3′-trailer sequences of the tRNA precursor (green). RNase T is a major processing enzyme in 3′-trailer removal. The resulting mature tRNA–CCA is repeatedly subjected to quality control (blue). RNase T removes the terminal A residue, resulting in tRNA–CC (end turnover). This truncated tRNA is then scrutinized by the CCA-adding enzyme. If the tRNA is intact, the enzyme completes the CCA end, releasing a functional tRNA. If the tRNA is damaged or unstable, the torque control mechanism of the CCA-adding enzyme leads to a refolding of the acceptor stem so that a second CCA triplet is added, and the tRNA is tagged by CCACCA for RNase R-mediated degradation (red). An occasional loss of the terminal A due to RNase T activity (dashed arrow) leads to a CCACC tag that is either reconverted into CCACCA (CCA-adding enzyme) or polyadenylated [poly(A) polymerase; not shown]. As a result, both products carry a 3′-tag long enough for being recognized by RNase R.