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The intrinsic structure of poly(A) RNA determines the specificity of Pan2 and Caf1 deadenylases

Nature Structural & Molecular Biology volume 26, pages 433–442 (2019)Cite this article

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An Author Correction to this article was published on 16 August 2019

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Abstract

The 3′ poly(A) tail of messenger RNA is fundamental to regulating eukaryotic gene expression. Shortening of the poly(A) tail, termed deadenylation, reduces transcript stability and inhibits translation. Nonetheless, the mechanism for poly(A) recognition by the conserved deadenylase complexes Pan2–Pan3 and Ccr4–Not is poorly understood. Here we provide a model for poly(A) RNA recognition by two DEDD-family deadenylase enzymes, Pan2 and the Ccr4–Not nuclease Caf1. Crystal structures of Saccharomyces cerevisiae Pan2 in complex with RNA show that, surprisingly, Pan2 does not form canonical base-specific contacts. Instead, it recognizes the intrinsic stacked, helical conformation of poly(A) RNA. Using a fully reconstituted biochemical system, we show that disruption of this structure—for example, by incorporation of guanosine into poly(A)—inhibits deadenylation by both Pan2 and Caf1. Together, these data establish a paradigm for specific recognition of the conformation of poly(A) RNA by proteins that regulate gene expression.

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Fig. 1: 3′ guanosines inhibit the Pan2 exonuclease.
Fig. 2: Ccr4–Not is inhibited by 3′ guanosines.
Fig. 3: Pan2 contacts the ribophosphate backbone of poly(A) RNA.
Fig. 4: Guanosines disrupt the conformation of oligo(A).
Fig. 5: Cytosines and uracils stack to form a helical conformation in the Pan2 active site.
Fig. 6: Nucleotide base stacking is required for Pan2 and Caf1 deadenylase activity.
Fig. 7: Model for the recognition of the intrinsic poly(A) structure by Pan2 and Caf1 deadenylases.

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Data availability

The structures generated during the current study have been deposited in the Worldwide Protein Data Bank under accession codes 6R9I (apo), 6R9J (A7-bound), 6R9M (AAGGAA-bound), 6R9O (AAGGA-bound), 6R9P (AAUUAA-bound) and 6R9Q (AACCAA-bound). Source data for Figs. 1b–e, 2b–d, 4c, 5a and 7b and Supplementary Figs. 1e,f, 2a, 6e–g and 7a,b are available in tabular form with the paper online. Source data for Figs. 1a, 2a and 6a–h are available in Supplementary Dataset 1 with the paper online. All annotated gels are available on Mendeley (https://doi.org/10.17632/zkfsh9nftk.1). All other data that support the findings of this study are available from the corresponding author upon reasonable request.

Change history

  • 16 August 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank C. Russo and M. Webster for assistance and advice; M. Yu and D. Ciziene for help with crystallography; M. Girbig for biGBac constructs; C. Johnson and S. McLaughlin for help with biophysics; and M. Egli, D. Brodersen, C. Oubridge, K. Nagai and R. Hegde for advice or comments on the manuscript. This work was supported by a Herchel Smith PhD Studentship from the University of Cambridge (to T.T.L.T.); the European Union’s Horizon 2020 research and innovation programme (ERC grant No. 725685, to L.A.P.); and the Medical Research Council, as part of United Kingdom Research and Innovation (MRC grant No. MC_U105192715, to L.A.P.). We acknowledge Diamond Light Source for beamtime (proposal No. MX15916), and the staff of beamlines I24, I03, I04 and I04-1 for assistance with crystal testing and data collection.

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  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Terence T. L. Tang, James A. W. Stowell, Chris H. Hill & Lori A. Passmore

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Contributions

T.T.L.T., J.A.W.S. and L.A.P. conceived the study. T.T.L.T. and J.A.W.S. purified proteins and prepared samples for crystallography. T.T.L.T., J.A.W.S. and C.H.H. collected crystallography data. T.T.L.T. built atomic models and interpreted the structure with help from C.H.H. T.T.L.T. carried out all other experiments. T.T.L.T. and L.A.P. wrote the manuscript. All authors discussed and commented on the final manuscript.

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Correspondence to Lori A. Passmore.

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Supplementary Figure 1 Inhibition of Pan2 by 3′ guanosines depends on their number and position.

a, Method for quantitation of deadenylation assays. 5′ 6-FAM-labeled (green star) poly(A) RNA substrates were incubated with the deadenylase. Reactions were quenched at fixed time intervals and products were visualized on a denaturing 20% polyacrylamide gel. The intensity of the band corresponding to intact RNA was quantified by densitometry. Intensities were normalized to the signal at time = 0 and plotted against time. Straight lines connect each point for clarity. Assays were carried out in triplicate and error bars represent standard deviation. b, Domain diagrams of proteins or protein complexes used in deadenylation assays. The pseudo-ubiquitin C-terminal hydrolase (UCH) and exonuclease (Exo) domains of Pan2 form a single structural unit12,13. PID, Pan3-interacting domain; ZnF, zinc finger; PK, pseudokinase; CC, coiled coil; CTD, C-terminal domain. c, Coomassie-stained SDS-PAGE of purified protein components used in deadenylation assays. The experiment for this gel has been carried out once. d, Denaturing RNA gels showing deadenylation by recombinant S. cerevisiae Pan2-Pan3 on RNA substrates containing guanosines in different positions and in different numbers at the 3′ end following the 30-adenosine poly(A) tail. These gels are representative of identical experiments performed 3 times. e-f, Analysis of deadenylation by S. cerevisiae Pan2-Pan3 (e) and Pan2UCH-Exo (f) on RNA substrates containing guanosines in different positions and in different numbers at the 3′ end following the poly(A) tail. Reactions were performed in triplicate (n = 3 independent experiments), the data points shown represent the mean, and error bars represent standard deviation. g, Two-color deadenylation assay by 50 nM recombinant S. cerevisiae Pan2UCH-Exo on 100 nM RNA substrates, which are differentially labeled on the 5′ end (Alexa-647-20mer-A30; 6-FAM-20mer-A14GA15). The experiment for this gel has been carried out once.

Source Data

Supplementary Figure 2 Crystallization of Pan2UCH-Exo E912A in complex with RNA.

a, Size exclusion chromatography of purified Pan2UCH-Exo E912A. The fractions outlined by dotted lines on the chromatogram (left) were pooled, concentrated, and used in crystallization. A Coomassie-stained gel of Pan2UCH-Exo E912A used in crystallization is shown (right). The purification and SDS-PAGE analysis of fractions thereof are representative of experiments which have been carried out 6 times. b, Deadenylation assay comparing the activity of Pan2UCH-Exo to the catalytic mutant Pan2UCH-Exo E912A. The 5′ 6-FAM-labeled RNA substrate consists of a 20mer non-poly(A) sequence followed by a poly(A) stretch of 30 adenosines. Deadenylation reactions were quenched at one-minute intervals and reaction products were visualized on a denaturing 20% polyacrylamide gel. For Pan2UCH-Exo E912A, the reaction was incubated over 24 h to demonstrate the lack of activity over the timescale of RNA soaking. These gels are representative of identical experiments performed 2 times. c, Crystals of Pan2UCH-Exo E912A. Crystals were obtained by an initial sparse matrix crystallization screen. The best diffracting condition was further subjected to optimization. d, Superposition of apo Pan2UCH-Exo E912A (blue) with the previously determined structure of apo Pan2UCH-Exo (grey, PDB: 4Q8H13). e, Superposition of apo Pan2UCH-Exo E912A (blue) with crystal structures containing bound RNA, including Pan2UCH-Exo E912A-A7 (green), Pan2UCH-Exo E912A-AAGGA (red), and Pan2UCH-Exo E912A-AAGGAA (orange). Superposition in (d) and (f) was carried out by the SSM tool in Coot55. Unmodeled loops are represented by dashed lines.

Source Data

Supplementary Figure 3 Interactions of Pan2UCH-Exo E912A with RNA.

a, Superposition of Pan2UCH-Exo-A7 (protein: blue, RNA: green) with S. pombe Caf1 (PDB: 3G0Z20; protein: pink). The structure of Caf1 contains two M2+ ions (pink) in the active site, which are shown as spheres. The numbers shown represent distance in Ångstroms. Relative to the scissile phosphate, the metal ions are approximately positioned for productive catalysis. b, Summary of interactions between Pan2UCH-Exo (blue) and A7 RNA (green), visualized using LigPlot+62.

Supplementary Figure 4 Docking of oligo(A) into Caf1 active site.

a, Surface representation of the Caf1 (PDB: 3G0Z20, pink) active site with a modelled poly(A) substrate (green). The RNA was modelled by superposition of Pan2UCH-Exo-A7 with the structurally homologous Caf1 by Coot55. Caf1 is colored by proximity to A7 from dark red (<3 Å) to pink (>7 Å). The Caf1 active site appears to contact both the ribophosphate backbone and adenine bases. b, Caf1 (pink) amino acid side chains which may contact the poly(A) substrate (green) in the docked model. Putative hydrogen bonds are indicated by dashed lines and interatomic distances are labeled in Ångstroms. The metal ions are shown as pink spheres. RNA placement was not refined and is therefore in a representative but likely non-optimal position.

Supplementary Figure 5 Guanosines disrupt the conformation of oligo(A) within the Pan2 active site.

a-b, Crystal structures of Pan2UCH-Exo E912A (cartoon, blue) bound to AAGGA (a) or AAGGAA (b), where the RNA is represented by red sticks. Unmodeled loops are represented by dotted lines. Inset: detailed views of the Pan2 active site. The electron density represents a feature enhanced map (FEM)56 contoured to 1.8σ. Nucleotides are numbered relative to the scissile phosphate bond, which is indicated by an arrow. c-d, Protein-RNA interactions between Pan2UCH-Exo E912A (blue) and AAGGA (c) or AAGGAA (d) (red). Hydrogen bonds are indicated by dashed lines and interatomic distances are labeled in Ångstroms.

Supplementary Figure 6 An aromatic residue at the base of the Pan2 active site is important for activity and specificity against guanosines.

a, Pan2 sequence alignment from representative eukaryotes. Sequences were aligned using the T-Coffee package59 and visualized using JalView60. Amino acids were colored by conservation using BLOSUM62. The conserved aromatic residue stacking against the RNA substrate is boxed in red. b, Coomassie-stained SDS-PAGE of Pan2UCH-Exo Y975A used in deadenylation assays. The experiment for this gel has been carried out once. c, Comparison of deadenylation by Pan2UCH-Exo with Pan2UCH-Exo Y975A on a 5′ 6-FAM-labeled (green star) RNA consisting of a 20mer non-poly(A) sequence followed by a poly(A) tail of 30 adenosines (20mer-A30). The Y975A mutant is much less active compared to the wild type. These gels are representative of identical experiments performed 2 times. d, Deadenylation by Pan2UCH-Exo on 5′ 6-FAM-labeled (green star) 20mer-A30 RNAs with three additional non-A nucleotides at the 3′ end where indicated. These gels are representative of identical experiments performed 3 times. e, Quantitation of deadenylation by Pan2UCH-Exo Y975A on poly(A) RNA substrates with different 3′ nucleotides. f, Comparison of deadenylation by Pan2UCH-Exo and Pan2UCH-Exo Y975A on 20mer-A30 and 20mer-A30G3. The horizontal dotted line indicates when half of RNA has been deadenylated, and the vertical dotted lines indicate the time at which half of RNA has been deadenylated for each condition. Terminal guanosines inhibit Pan2UCH-Exo Y975A ~11-fold (8.5 h for 20mer-A30G3 compared to 0.8 h for 20mer-A30), compared to a ~117-fold inhibition (3.9 h for 20mer-A30G3 compared to ~2 min for 20mer-A30) of Pan2UCH-Exo. g, Comparison of deadenylation by Pan2UCH-Exo Y975A and the catalytic mutants Pan2UCH-Exo E912A and Pan2UCH-Exo E912A Y975A, as well as RNA incubated alone. The negative control demonstrates that Pan2UCH-Exo Y975A retains deadenylase activity greater than background RNA degradation. Thus, the aromatic residue against which the substrate π-stacks is important for the activity of Pan2 and contributes to nucleotide specificity. For panels e-g, the experiments were carried out in triplicate (n = 3 independent experiments), the data points shown represent the mean, and the error bars represent standard deviation.

Source Data

Supplementary Figure 7 The structure of oligo(A) is disrupted by guanosines in both 5′ and 3′ directions.

a, Circular dichroism spectra of 9.0 µM A5 (green) and AAGGA RNAs as averages from 9 scans of the same sample obtained at 0.5 nm intervals. These spectra are representative of identical experiments performed 2 times. b, Circular dichroism spectra of G2A8 (red), A8G2 (orange), A6G2A2 (pink), A2G2A6 (blue), and A4G2A4 (purple) RNAs, compared to A10 RNA (green). The RNAs are present at 25.0 µM. Each curve represents an average from 9 scans of the same sample obtained at 0.5 nm intervals. These spectra are representative of identical experiments performed 2 times.

Source Data

Supplementary Figure 8 Poly(A) stacking is important for deadenylation by Pan2-Pan3.

a-b, Deadenylation by S. cerevisiae Pan2-Pan3 of 5′ 6-FAM-labeled (green star) RNAs consisting of a 20mer non-poly(A) sequence followed by a tail of the indicated sequence. RNAs either had no additional nucleotides (a) or guanosines (b) in the middle of the poly(A) tail. Red asterisks indicate the point of inhibition. c, Determination of site of inhibition of S. cerevisiae Pan2-Pan3. Deadenylation reactions of 5′ 6-FAM-labeled (green star) 20mer-A14GGA14 by S. cerevisiae Pan2-Pan3 were repeated and the point of inhibition determined by comparing the reaction at 33 minutes to an alkaline hydrolyzed RNA marker. Inhibition began when Pan2-Pan3 reached a 3′ sequence of –GGAAAA. Individual bases of the marker are indicated. The dashed line separates lanes which have different contrast adjustments to visualize the fainter bands in the marker lane. This gel is representative of identical experiments performed 2 times. d, The chemical structures of uridine and dihydrouridine. e-f, Deadenylation by S. cerevisiae Pan2-Pan3 of 5′ 6-FAM-labeled (green star) RNAs consisting of a 20mer non-poly(A) sequence followed by a tail of the indicated sequence. RNAs either had two uracils (e), or dihydrouracils (abbreviated D, panel f) in the middle of the poly(A) tail. Red asterisks indicate the point of inhibition. These gels are representative of identical experiments performed 2 times. g, Determination of site of inhibition of S. cerevisiae Pan2-Pan3. Deadenylation reactions of 5′ 6-FAM-labeled (green star) 20mer-A14DDA14 by S. cerevisiae Pan2-Pan3 were repeated and the point of inhibition determined by comparing the reaction at 33 minutes to an alkaline hydrolyzed RNA marker. Inhibition began when Pan2-Pan3 reached a 3′ sequence of –DDAAAA. Individual bases of the marker are indicated. The dashed line separates lanes which have different contrast adjustments to visualize the fainter bands in the marker lane. This gel is representative of identical experiments performed 2 times.

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Supplementary Information

Integrated Supplementary Figures 1–8 and Supplementary Dataset 1

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Supplementary Video 1

Crystal structure of Pan2UCH-Exo bound to A7 RNA. The crystal structure of Pan2UCH-Exo bound to A7 RNA is shown. The UCH and exonuclease domains are in blue and the RNA is in green. The electron density map shown corresponds to a feature-enhanced map contoured to 1.3σ.

Supplementary Video 2

Crystal structure of Pan2UCH-Exo bound to AAGGA RNA. The crystal structure of Pan2UCH-Exo bound to AAGGA RNA is shown. The UCH and exonuclease domains are in blue and the RNA is in red. The electron density map shown corresponds to a feature-enhanced map contoured to 1.3σ.

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Tang, T.T.L., Stowell, J.A.W., Hill, C.H. et al. The intrinsic structure of poly(A) RNA determines the specificity of Pan2 and Caf1 deadenylases. Nat Struct Mol Biol 26, 433–442 (2019). https://doi.org/10.1038/s41594-019-0227-9

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  • DOI: https://doi.org/10.1038/s41594-019-0227-9

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