tRNAs marked with CCACCA are targeted for degradation

Abstract

The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking structurally unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. In addition, hypomodified mature tRNAs are subjected to CCACCA addition as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addition is a universal mechanism for controlling tRNA levels and preventing errors in translation.

Fig. 1. CCACC(A) is added to the MEN β tRNA-like small RNA

(A) mascRNA and the MEN β tRNA-like small RNA are predicted to fold into cloverleaf secondary structures. The mouse homologs are shown. 3′ RACE revealed that different 3′ terminal sequences are post-transcriptionally added to the RNAs (shown in red). (B) Northern blot analysis using 15 μg of total RNA from five cell lines.

Fig. 2. An unstable acceptor stem is isomerized to allow for CCACCA addition

(A) Chimeric transcripts generated. (B and C) Wild-type mascRNA (denoted WT) and the chimeras were incubated in vitro with human (B) or S. shibatae (C) CCA-adding enzyme. [α-³²P] CTP or [α-³²P] ATP was added to assay for incorporation of each nucleotide into the unlabeled substrates. In the upper panels, substrates lacked CCA so all substrates should undergo, at minimum, one round of CCA addition, with a second round of CCA addition (yielding CCACCA) generating a 3 nucleotide shift in mobility. In the lower panels, CCA was already present so only RNAs that are CCACCA targets yield visible bands. (D) WT mascRNA or chimeras in which the acceptor stem was swapped with that of a MEN β homolog were used as substrates. Mismatches and G-U wobble base pairs are in red and blue, respectively. (E) Model for how CCACCA is added to the mouse MEN β tRNA-like small RNA.

Fig. 3. Destabilizing tRNAs causes CCACCA addition and transcript degradation in vitro

(A) Pie charts showing the first two nucleotides of canonical tRNAs from human (representing 631 tRNAs), E. coli (88 tRNAs), and S. solfataricus (46 tRNAs). (B) tRNA^Arg(TCG) transcripts containing point mutations were used as in vitro substrates for the E. coli CCA-adding enzyme. (C) Radiolabeled wild-type or mutant arginine tRNAs ending in CCA or CCACCA, respectively, were incubated in HeLa nuclear extracts for the indicated times. Arrow denotes the accumulation of wild-type tRNAs cleaved in the anticodon loop.

Fig. 4. CCACCA is added to tRNAs being actively degraded in vivo

(A) The percentage of tRNA^Ser(CGA) and tRNA^Ser(UGA) transcripts ending in an extended CCA motif in yeast strains grown at 28°C (designated 0 hr) or 37°C for the indicated times was determined. The structure of tRNA^Ser(CGA) is shown. (B) The endogenous tRNA^Ser(CGA) gene was replaced in the trm44-Δ tan1-Δ strain with the variants shown and the strains were subjected to 3′ RACE PCR. Error bars represent standard deviation. (C) A serine tRNA ending in CCACCA was degraded in vitro by yeast Rrp44. The Rrp44 D551N mutant lacks exonuclease catalytic activity. (D) Yeast Rrp44 and Xrn1 cooperate to degrade tRNAs ending in CCACCA in vitro. The enzymes were titrated to identify conditions in which minimal degradation was observed when only a single exonuclease was present.