The human cap-binding complex is functionally connected to the nuclear RNA exosome

Abstract

Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC–ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC–NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5′ cap links transcription termination to exosomal RNA degradation through CBCN.

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Figure 1: AC of NEXT-complex components hMTR4 and RBM7 reveals stoichiometrically interacting ZC3H18 and CBCA complex.
Figure 2: AC of CBCA complex components CBP80, CBP20 and ARS2 reveals stoichiometric CBCN complex.
Figure 3: AC of ZC3H18 identifies stoichiometric CBCN complex.
Figure 4: CBCN components share common RNA targets.
Figure 5: Hyperaccumulation of PROMPTs upon co-depletion of CBCA with components of NEXT or RNA-exosome complexes.
Figure 6: CBCA depletion causes inefficient transcription termination and exosome-dependent removal of read-through transcripts.
Figure 7: CBCA depletion displays a genome-wide termination defect of PROMPT transcription.

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Acknowledgements

We thank M. Schmid, S. Lykke-Andersen, M. Lubas, A. Dziembowski and D. Libri for critical comments to the manuscript. Special thanks go to E. Marchal for excellent technical assistance. E. Izaurralde (Max Planck Institute for Developmental Biology) and J. Lykke-Andersen (Division of Biological Sciences at University of California, San Diego) are acknowledged for sharing reagents. This work was supported by the Danish National Research Foundation (grant DNRF58), the Danish Cancer Society and the Lundbeck and Novo Nordisk Foundations (T.H.J.); the US National Institutes of Health (grant U54 GM103511, to M.P.R.), the l'ARC and La Ligue Contre Le Cancer (E.B.), the Foundation for Strategic Research (grant FFL09-0130, to R.S.) and the European Community's Seventh Framework Programme (FP7/2007-2013) under grant no. 241548 (MitoSys, to A.H.).

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Contributions

M.D. and J.B. performed ACMS experiments and analyzed the data together with J.S.A., J.L. and M.P.R.; I.P. and A.H. contributed tagged cell lines. C.V., M.H. and E.B. performed and analyzed RIP experiments. P.R.A., M.S.K. and A.S. performed experimental RNA analyses. P.R.A. and E.N. performed and analyzed ChIP experiments. P.R.A., H.S. and R.S. analyzed RNA-seq data. P.R.A., M.D., M.S.K. and T.H.J. wrote the manuscript.

Corresponding author

Correspondence to Torben Heick Jensen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of near-endogenous expression levels of tagged proteins.

Western blotting analyses of tagged proteins used for AC/MS analyses as well as their endogenous counterparts. Membranes were probed with antibodies targeting the indicated factors. Tagged versions of CBP20, CBP80 and ZC3H18 were expressed at endogenous levels, while tagged versions of ARS2 and hMTR4 were expressed at lower levels than their endogenous versions. RBM7-LAP was ~2-fold overexpressed, which is unlikely to have caused any artificial interactions due to the high level of congruency between AC/MS analyses of RBM7-LAP, hMTR4-LAP and ZCCHC8-FLAG6

Supplementary Figure 2 Co-depletion of major 5'-3' exonucleolytic enzymes does not affect PROMPT levels.

(a) Western blotting analysis of whole cell extracts showing protein depletion upon the indicated siRNA administrations. Membranes were probed with the indicated antibodies. Anti-actin antibody was used as a loading control. (b) XRN1 + XRN2-depletion does not increase PROMPT levels of exosome-depleted cells. Levels of the indicated PROMPTs were measured and displayed as in Fig. 5b.

Supplementary Figure 3 Effect of depletion of CBCA components on transcription termination of the U1- and U8-encoding genes.

(a) Transcriptional read-through analysis of the U1 gene upon the indicated factor depletions and displayed as in Fig. 6b. (b) Northern blotting analysis of total RNA purified from HeLa cells subjected to the indicated siRNA transfections (depletion levels shown in Fig. 5a). Position of the utilized 5'end radiolabeled probe to primarily detect 3'extended U8 RNA species is shown at the top. Factor depletions (Fig. 5a), data display and control were done as in Fig. 5c. (c) Transcriptional read-through analysis of the U8 gene upon the indicated factor depletions and displayed as in Fig. 6b.

Supplementary Figure 4 Mean coverage ratios around TSSs upon CBCN factor depletion.

Plots are displayed similarly to those in Figure 7. (a) PROMPT accumulation upon combinatorial depletion of hRRP40 and CBCN relative to control siRNA. (b) PROMPT accumulation upon combinatorial depletion of ZCCHC8 and CBCN relative to control siRNA. (c) PROMPT accumulation upon combinatorial depletion of ZCCHC8 and CBCN relative to ZCCHC8 depletion. Faded areas represent 95% confidence intervals for each curve.

Supplementary Figure 5 Mean coverage ratios around histone mRNA TSSs upon CBCN factor depletion.

Plots are displayed similarly to those in Fig. 7. For a-d upper panels display the region around annotated 3'ends of mature replication-dependent histone (RDH) genes, while lower panels display that of replication-independent histone (RIH) genes. (a) Mean coverage as tags per event (TPE) at histone gene TTSs. (b) Histone RNA accumulation upon single factor depletion relative to control siRNA. (c) Histone RNA accumulation upon combinatorial depletion of hRRP40 and CBCN relative to control siRNA. (d) Histone RNA accumulation upon combinatorial depletion of hRRP40 and CBCN relative to hRRP40 depletion.

Supplementary Figure 6 Uncropped gel images of northern blots in Figure 5 and Supplementary Figure 3.

(a) Uncropped image of the northern blot shown in Fig. 5c. (b) Uncropped image of the northern blot shown in Supplementary Fig. 3b.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1 and 8–13 and Supplementary Note (PDF 1893 kb)

Supplementary Table 2

hMTR4-LAP ACMS (XLSX 36 kb)

Supplementary Table 3

RBM7-LAP ACMS (XLSX 17 kb)

Supplementary Table 4

CBP80–3× FLAG ACMS (XLSX 23 kb)

Supplementary Table 5

CBP20–3× FLAG ACMS (XLSX 17 kb)

Supplementary Table 6

LAP-ARS2 ACMS (XLSX 16 kb)

Supplementary Table 7

ZC3H18–3× FLAG ACMS (XLSX 22 kb)

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Andersen, P., Domanski, M., Kristiansen, M. et al. The human cap-binding complex is functionally connected to the nuclear RNA exosome. Nat Struct Mol Biol 20, 1367–1376 (2013). https://doi.org/10.1038/nsmb.2703

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