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. 2015 Feb 12;160(4):644-658.
doi: 10.1016/j.cell.2015.01.005. Epub 2015 Jan 29.

On-enzyme Refolding Permits Small RNA and tRNA Surveillance by the CCA-adding Enzyme

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Free PMC article

On-enzyme Refolding Permits Small RNA and tRNA Surveillance by the CCA-adding Enzyme

Claus-D Kuhn et al. Cell. .
Free PMC article

Abstract

Transcription in eukaryotes produces a number of long noncoding RNAs (lncRNAs). Two of these, MALAT1 and Menβ, generate a tRNA-like small RNA in addition to the mature lncRNA. The stability of these tRNA-like small RNAs and bona fide tRNAs is monitored by the CCA-adding enzyme. Whereas CCA is added to stable tRNAs and tRNA-like transcripts, a second CCA repeat is added to certain unstable transcripts to initiate their degradation. Here, we characterize how these two scenarios are distinguished. Following the first CCA addition cycle, nucleotide binding to the active site triggers a clockwise screw motion, producing torque on the RNA. This ejects stable RNAs, whereas unstable RNAs are refolded while bound to the enzyme and subjected to a second CCA catalytic cycle. Intriguingly, with the CCA-adding enzyme acting as a molecular vise, the RNAs proofread themselves through differential responses to its interrogation between stable and unstable substrates.

Figures

Figure 1
Figure 1. Catalysis Is Crucial to Detect Unstable RNAs
(A) G70 of the arginyl-tRNATCG minihelix was mutated to A, resulting in the miniUR transcript that has significant structural instability within its acceptor stem. The archaeal CCA-adding enzyme is shown in cartoon representation with the head domain in purple, the neck domain in green, the body domain in blue and the tail domain in cyan. Bound RNA is shown in atom colors with oxygens in red, nitrogens in blue, phosphorous in orange and carbons in light blue. 2Fo-Fc density for the bound unstable RNA is contoured at 1 σ throughout Figure 1. (B) Close-up view of the acceptor stem. (C) Close-up view of the acceptor stem following CTP addition (miniUR-C+CTP). (D) MiniUR before and after CTP addition. Nucleotides labeled in black have similar positions in both structures, the mutation is boxed in red. (E) Comparison of the CCA-adding enzyme in complex with a wild-type tRNA minihelix (miniR) (PDB code 2DR5) (Tomita et al., 2006) or in complex with the unstable miniUR minihelix after CTP addition. See also Figure S1.
Figure 2
Figure 2. Clockwise Screw Motion and RNA Compression upon Active Site Closure Structural transitions upon active site closure
(A) RNA and protein domains of the open complex (miniUR-CCAC) are shown in cartoon representation in gray. The preinsertion complex (miniUR-CCAC+CTP) is colored as in Figure 1A with miniUR-CCAC+CTP RNA, including the incoming CTP, in orange. Catalytic magnesium ions are in magenta. The clockwise screw motion of the head domain and RNA is indicated. (B) Close-up view of the acceptor stem of miniUR-CCAC (gray) and miniUR-CCAC+CTP (orange). The A70 mutation is boxed in red. RNA compression is indicated by an arrow. (C) Top-down view of (B) including the miniUR-CCAC+CTP β-hairpin (purple). Clockwise RNA rotation is indicated. (D) Location of the RNA bulge in all three preinsertion complex structures of the second CCA cycle. Common nucleotides to all structures are numbered in black, unique ones follow the RNA color scheme. Loops 1 and 2 are shown for miniUR-CCAC+CTP only. (E) In vitro CCA-addition assays were performed to assay the ability of AfCCA containing Loop 1 insertions to incorporate [α-32P] ATP onto stable or unstable (G70A) arginyl-tRNATCG minihelices ending in -C. The insertion of three or six glutamates abolished the ability of the CCA-adding enzyme to mark unstable RNAs for degradation by adding CCACCA. See also Figure S2 and Movies S1 and S2.
Figure 3
Figure 3. Unstable RNA Is Held onto the CCA-Adding Enzyme through Ionic “Tweezers”
(A) Structure of the adenosine preinsertion complex of the Menβ tRNA-like small RNA (miniMβ-CCACC+AMPcPP), as in Figure 1A. The disordered RNA bulge is dashed and colored in pink. (B) Comparison between the miniMβ-CCACC+AMPcPP and miniUR-CCACC+AMPcPP RNA structures. Both RNAs are schematically diagrammed on the right with disordered nucleotides in gray. The kink in miniMβ-CCACC RNA is sketched. (C) Temperature factor distribution of miniMβ-CCACC+AMPcPP. Low temperature factors of 20 Å2 are in blue, intermediate values in yellow, and values above 80 Å2 in red. (D) Electrostatic surface potential of the archaeal CCA-adding enzyme. Blue depicts positively charged, white neutral, and red negatively charged areas. The two positively charged patches holding the acceptor stem are indicated. MiniMβ RNA is in cartoon and colored in pink. The close-up view details the ionic interactions that glue the top of the acceptor stem to the enzyme. A schematic of the “ionic tweezers” is shown. (E) In vitro CCA-addition assays using wild-type or mutant AfCCA and the unstable (G70A) arginyl-tRNATCG minihelix (miniUR) substrate. Weakening the strong interactions between the RNA 5′-end and the enzyme impaired the second CCA-addition cycle, whereas the first cycle was unaltered. See also Figure S3.
Figure 4
Figure 4. On-Enzyme RNA Refolding Permits the Transition from the First to the Second CCA Cycle
(A) The two interchangeably bound RNA conformations observed in bromouridine (BrU)-labeled miniUR-CCACCA co-crystals. BrU and RNA instabilities are highlighted in red. (B) The double-helical part of the non-refolded RNA from (A) is shown in cartoon representation and colored in gray. BrU are in red. The anomalous difference Fourier map is colored in green and contoured at 3.5 σ. (C) Structure of the full-length human mitochondrial CCA-adding enzyme. Protein domains are colored according to the archaeal enzyme in Figure 1A. The newly resolved tail domain is highlighted. A tRNA minihelix is modeled according to PDB code 1VFG (Tomita et al., 2004). An ethylthio-purine crosslinker was incorporated at tRNA position C56 and crosslinked to the CCA-adding enzyme carrying a G379C mutation. (D) Silver-stained SDS-PAGE of the crosslinked human CCA-adding enzyme and the ethylthio-purine-derivatized minihelices. Crosslinked complexes show retarded gel mobility, with the G379C and G364C mutants showing the highest crosslinking efficiency. (E) In vitro CCA addition assays with crosslinked complexes. The wild-type and G70A mutant arginyl-tRNATCG minihelices are denoted as stable and unstable, respectively. Reactions 2–5 were under oxidizing, whereas reactions 6–10 were under reducing conditions. Radioactive nucleotides are indicated above the lanes. Unstable minihelices that had never been crosslinked were used as controls (reactions 9–10). (F) In vitro competition experiments. In lanes 1–4, full-length arginyl-tRNATCG (G70A) was pre-bound to the human and archaeal CCA-adding enzyme in the presence of 2 µM CTP before adding cold ATP supplemented with [α32P]-ATP and varying amounts of the hMENβ transcript (up to a 20-fold excess). In lanes 5–8, both RNAs were added simultaneously along with ATP. An equal amount of arginyl-tRNATCG was added in all lanes, whereas hMENβ amounts varied as denoted at the top. Pre-bound RNA was efficiently extended to CCACCA irrespective of added excess RNA, indicating that the bound RNA refolds “on-enzyme.” When added simultaneously, both RNAs compete for binding to the CCA-adding enzyme. See also Figure S4.
Figure 5
Figure 5. Active Site Closure Gauges RNA Stability and Triggers Refolding
(A) The intermediate adenosine preinsertion complex of Menβ (miniMβ-CCACC+AMPcPP-i) shown as in Figure 1A. Residues contributed by the head domain are in atom colors with carbons in purple. The disordered residues Ala95 and Asp96 are dashed in purple. Arg224 from the neck domain is in atom colors with carbons in green. A dashed circle highlights the missing Metal B. Improper positioning of the incoming nucleotide is shown with black dashes. (B) Superposition of the adenosine intermediate and preinsertion complexes (miniMβ-CCACC+AMPcPP). The intermediate state RNA and AMPcPP are in gray, while the intermediate β-hairpin and Asp110 are in purple. Intermediate Arg224 is in green. The entire preinsertion complex (miniMβ-CCACC+AMPcPP) is in pink. Three proofreading interactions, the AMPcPP base edge recognition and the Asp110 general base contact to the 3′-hydroxyl are shown with green dashed lines for the preinsertion complex, as is Metal B bound to AMPcPP. (C) Comparison of the miniMβ-CCACC+AMPcPP complex and the termination complex of the first CCA cycle (PDB code 1SZ1) (Xiong and Steitz, 2004). The incoming AMPcPP is depicted for Menβ. Canonical tRNA numbering is used for Menβ to simplify comparison. The active site residues that the RNA stacks against in the termination complex are shown as sticks. The counter-clockwise twist between the Menβ preinsertion complex and the tRNA termination complex is indicated. (D) In vitro CCA addition assays with full-length stable and unstable (G70A) arginyl-tRNATCG and either the human or the archaeal CCA-adding enzyme. Cold nucleotide concentrations (ATP+CTP) were increased in the presence of radioactive CTP. CCA tail length is indicated. See also Figure S5 and Movies S3 and S4.
Figure 6
Figure 6. Model of RNA Surveillance by the CCA-Adding Enzyme
The head and neck domains of the CCA-adding enzyme are shown as abstract bodies colored in purple and green, respectively, with the β-hairpin as an oval. Bound unstable RNA is shown in orange with its compression state changing during the CCA-addition cycles. Steps 1–10 are described in the text. See also Figure S6.

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