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CBC–ARS2 stimulates 3′-end maturation of multiple RNA families and favors cap-proximal processing

Nature Structural & Molecular Biology volume 20, pages 1358–1366 (2013)Cite this article

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Abstract

The nuclear cap–binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small, capped RNAs such as pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, ARS2 and PHAX, and show that both CBCAP and CBC–ARS2 complexes can be reconstituted from recombinant proteins. ARS2 stimulates PHAX binding to the CBC and snRNA 3′-end processing, thereby coupling maturation with export. In vivo, CBC and ARS2 bind similar capped noncoding and coding RNAs and stimulate their 3′-end processing. The strongest effects are for cap-proximal polyadenylation sites, and this favors premature transcription termination. ARS2 functions partly through the mRNA 3′-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. ARS2 is thus a major CBC effector that stimulates functional and cryptic 3′-end processing sites.

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Figure 1: The CBCAP complex contains PHAX, CBP20, CBP80 and ARS2.
Figure 2: The CBCAP complex can be reconstituted in vitro, and ARS2 contributes to the association of PHAX with CBC in vivo.
Figure 3: Identification of RNAs bound by the CBC and ARS2.
Figure 4: CBC and ARS2 are involved in 3′-end processing and transcription termination of snRNA genes.
Figure 5: Knockdowns of CBC and ARS2 lead to readthrough of the 3′ processing site of nonpolyadenylated histone mRNAs and polyadenylated mRNAs.
Figure 6: CBC and ARS2 associate with the cleavage factor CLP1.
Figure 7: CBC and ARS2 preferentially stimulate cap-proximal sites.

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Acknowledgements

We thank the Functional Proteomics Platform (FPP, Montpellier Languedoc-Roussillon, France) for the use of their instruments. We thank I. Poser (Max Planck Institute Dresden) for the gift of the CLP1-GFP BAC cell line and H. Le Hir (École Normale Supérieure, Paris) and M.S. Kristiansen (University Aarhus) for critical reading of the manuscript. D.L. was supported by a fellowship from the Fondation pour la Recherche Médicale, M.H. was supported by the French Ministère de la Recherche et de la Technologie, and F.P. was supported by La Ligue Nationale Contre le Cancer (LNCC). This work was funded by grants from the Danish National Research Foundation (grant DNRF58) to T.H.J. and from the French Centre National de la Recherche Scientifique, the Association de la Recherche contre le Cancer and the LNCC to E.B.

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  1. Frédéric Pontvianne & Daniela Lener

    Present address: Present addresses: Laboratoire Génome et Développement des Plantes, Centre National de la Recherche Scientifique, Perpignan, France (F.P.) and Institut de Génétique et de Biologie Moléculaire et Cellulaire–Centre National de la Recherche Scientifique, Strasbourg, France (D.L.).,

Authors and Affiliations

  1. Equipe labellisée Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier–Centre National de la Recherche Scientifique, Montpellier, France

    Marie Hallais, Frédéric Pontvianne, Daniela Lener, Nour El Houda Benbahouche, Thierry Gostan, Marie-Cécile Robert, Céline Verheggen & Edouard Bertrand

  2. Department of Molecular Biology and Genetics, Centre for mRNP Biogenesis and Metabolism, Aarhus University, Aarhus, Denmark

    Peter Refsing Andersen & Torben Heick Jensen

  3. European Molecular Biology Laboratory Grenoble Outstation, Grenoble, France

    Marcello Clerici & Stephen Cusack

  4. Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Montpellier, France

    Franck Vandermoere

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Contributions

M.H., F.P., P.R.A., M.C., D.L., N.E.H.B., M.-C.R., C.V. and E.B. performed the experiments, T.G. and E.B. analyzed the microarray data, F.V. analyzed the proteomic experiment, all authors participated in the conception of the experiments, S.C. supervised the in vitro reconstitution experiment, T.H.J. supervised the ChIP experiment, and E.B. and C.V. wrote the manuscript and supervised the work.

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Correspondence to Céline Verheggen or Edouard Bertrand.

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Integrated supplementary information

Supplementary Figure 1 Characterization of the CBCAP complex.

(A) Characterization of anti-PHAX monoclonal antibodies by Western blotting. Samples were from Hela cells treated with siRNAs against PHAX or FFL as control, and analyzed by Western blots using the indicated antibody. (B) GFP-Ars2 is not recruited to LacO arrays by KPNA2-Laci. Micrographs are from U2OS cells bearing a large LacO repeat, co-transfected with GFP-Ars2 and KPNA2-Laci-mCherry and observed by fluorescence microscopy. (C) Protein-protein interactions within the CBCAP complex analyzed by two-hybrid tests. (D) Alignments and domains of human, drosophila, plant and fission yeast Ars2. Schematics indicate putative functional domains of human Ars2.

Supplementary Figure 2 Characterization of the Flp-In cell lines.

(A) Characterization of 3x-Flag-Ars2 and 3xFlag-CBP20 Hela Flip-In cells by Western blotting. Samples are lysates from 3x-Flag-Ars2 and 3xFlag-CBP20 Flp-In cells analyzed by Western blotting with anti-Flag, anti-Ars2 and anti-GAPDH antibodies as indicated. Arrows: the Flag-tagged protein. Stars: non-specific binding of the antibodies. Ct: control parental cell line. (B) Efficiency of 3xFlag immunoprecipitation analyzed by Western blotting. Samples are lysates (Input) of 3xFlag-Ars2 Flp-In cells (Ars2) or control cells (Ct), expressing 3x-Flag-Ars2, immunopurified with anti-Flag antibodies (IP), and analyzed with anti-Flag antibodies. Sup: supernatants from the IP; W: last wash of the beads. Input, Sups, W: 10% of pellet. (C) Comparison of the enrichment of capped and uncapped non-coding RNAs in the various IPs. Values are enrichment fold from the microarray analysis. Control IP is from parental Flp-In cells that express no tagged protein.

Supplementary Figure 3 Identification and characterization of the RNAs present in the CBP20 and Ars2 Flag IPs.

(A) Analysis of the RNAs enriched in one IP but not in the other. For an IP “X”, the list of RNA enriched in the IP “X” (value greater than mean plus 1,5 STD) but not in the IP “Y” (value lower than mean plus 1,5 STD) was established, and the number represent the enrichment of these RNAs in the IP “Y”. In parenthesis: p Values. (B) Enrichment of specific RNA families in the various IPs. Numbers represent the mean enrichment value for the indicated family, calculated from the microarray data (Log2[enrichment of the specific IP/control IP]). In parenthesis: p Values.

Supplementary Figure 4 Depletion of CBC and Ars2 induces readthrough of U11 and U3 processing sites.

(A) Ars2 depletion and tiling array analysis of RNAs. Samples are from Hela cells treated with Ars2 or control siRNAs, analyzed by tiling microarrays using random-primed libraries (biological triplicates). The graph corresponds to the region of the U11 gene. Transcription from left to right. Bottom: chromosome coordinates. (B) Readthrough assay for U3 terminator and luciferase activity. Samples are from lysates of Hela cells transfected with a U3 plasmid and the indicated siRNAs, and values represent Firefly luciferase activity expressed as ratio over a co-transfected Renilla reporter plasmid.

Supplementary Figure 5 Depletion of CBC and Ars2 induces readthrough of CNIH4 and NR3C1 poly(A) sites.

(A) Read-through assays for of the CNIH4 polyA site and luciferase activity. Samples are lysates from Hela cells transfected with a CNIH4-luciferase plasmid and the indicated siRNAs, and values represent Firefly luciferase activity expressed as ratio over a co-transfected Renilla reporter plasmid. (B) Schematic of the NR3C1 cryptic polyA region. Four potential AAUAAA-like hexamers, with the mutants generated, are depicted. (C) Read-through assays for the NR3C1 polyA region and measurments of RNA levels by RT-qPCR. Samples are from Hela cells transfected with the indicated plasmid and siRNAs, and values represent readthrough RNA levels expressed as ratio of an upstream amplicon, measured by RT-qPCR.

Supplementary Figure 6 Association of CLP1 with RNA polymerase II and uncropped gels of main figures.

(A) CLP1 co-precipitation with RNA Pol II and Western blot analysis. Samples are lysates from Hela cells transfected with a vector expressing GFP-CLP1, immunoprecipitated with anti-GFP antibodies, and analyzed by Western blotting with an antibody against RBP1 (the largest RNA pol II subunit). Input: 5% of pellets. (B-C) Uncropped gels of Figure 1B. (D-E) Uncropped gels of Figure 6B. (F-G) Uncropped gels of Figure 6C.

Supplementary Figure 7 Efficiency of siRNA treatments and P values of luciferase assays.

(A) Samples are lysates from HeLa cells transfected with the indicated siRNAs and analyzed by Western blots with the indicated antiodies. Stars: non-specific bands used as loading controls. (B) p Values of the luciferase assays shown in Figure 7A. Values represent probabilities calculated with two-sided t-tests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 2 (PDF 5905 kb)

Supplementary Table 1

Excel spreadsheet containing the ‘all genes’ microarray/IP data, as well as the results of the GFP-CLP1 SILAC proteomic experiment (XLSX 591 kb)

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Hallais, M., Pontvianne, F., Andersen, P. et al. CBC–ARS2 stimulates 3′-end maturation of multiple RNA families and favors cap-proximal processing. Nat Struct Mol Biol 20, 1358–1366 (2013). https://doi.org/10.1038/nsmb.2720

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