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DGCR8 HITS-CLIP reveals novel functions for the Microprocessor

Abstract

The Drosha–DGCR8 complex (Microprocessor) is required for microRNA (miRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as the endonuclease. Using high-throughput sequencing and cross-linking immunoprecipitation (HITS-CLIP) we identified RNA targets of DGCR8 in human cells. Unexpectedly, miRNAs were not the most abundant targets. DGCR8-bound RNAs also comprised several hundred mRNAs as well as small nucleolar RNAs (snoRNAs) and long noncoding RNAs. We found that the Microprocessor controlled the abundance of several mRNAs as well as of MALAT1. By contrast, DGCR8-mediated cleavage of snoRNAs was independent of Drosha, suggesting the involvement of DGCR8 in cellular complexes with other endonucleases. Binding of DGCR8 to cassette exons is a new mechanism for regulation of the relative abundance of alternatively spliced isoforms. These data provide insights in the complex role of DGCR8 in controlling the fate of several classes of RNAs.

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Figure 1: CLIP for endogenous and overexpressed DGCR8 in HEK 293T cells.
Figure 2: DGCR8 binds and controls the stability of the long intergenic noncoding RNA MALAT1.
Figure 3: Binding of DGCR8 to snoRNAs affects their abundance independently of Drosha.
Figure 4: Microprocessor binding to mRNAs.
Figure 5: Binding of DGCR8 to cassette exons can modulate the relative abundance of alternatively spliced isoforms.

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Acknowledgements

We are grateful to S. Heras for discussions and critical reading of the manuscript; N. Kim (Seoul National University) for Flag-Drosha, dominant negative DGCR8 and Drosha expression vectors; B. Seraphin (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg) for the Dcp1 antibody and R. Blelloch (University of California, San Francisco) for Dicer knockout and Dicer flox/flox cell lines. This work was supported by the Medical Research Council and by the Wellcome Trust (084057/Z/07/Z to J.F.C.; A.S., G.M. and S.M. were partially funded by this). E.E. and M.P. were supported by grants from the Spanish Ministry of Science and by the Sandra Ibarra Foundation (BIO2008-01091, BIO2011-23920 and CSD2009-00080). S.M. was the recipient of a European Molecular Biology Organization long-term postdoctoral fellowship. M.P. is supported by the Novo Nordisk Foundation. J.F.C is a recipient of a Wellcome Trust Senior Investigator Award (grant 095518/Z/11/Z).

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S.M. and J.F.C. conceived, designed and interpreted the experiments. S.M., G.M. and A.S. performed experiments and analyzed data. M.P. and E.E. performed all bioinformatics analysis, including mapping of the CLIP tags to the genome and statistical analysis. J.F.C. supervised the project. All authors wrote the manuscript.

Corresponding author

Correspondence to Javier F Cáceres.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1,3 and Supplementary Note (PDF 3023 kb)

Supplementary Table 2

List of alternative splicing changes in Dgcr8 knockout mouse embryonic stem cells. (XLS 1187 kb)

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Macias, S., Plass, M., Stajuda, A. et al. DGCR8 HITS-CLIP reveals novel functions for the Microprocessor. Nat Struct Mol Biol 19, 760–766 (2012). https://doi.org/10.1038/nsmb.2344

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