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The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a

Abstract

hnRNP A1 is an RNA-binding protein involved in various aspects of RNA processing. Use of an in vivo cross-linking and immunoprecipitation protocol to find hnRNP A1 RNA targets resulted in the identification of a microRNA (miRNA) precursor, pre-miR-18a. This microRNA is expressed as part of a cluster of intronic RNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92, and potentially acts as an oncogene. Here we show that hnRNP A1 binds specifically to the primary RNA sequence pri-miR-18a before Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity with pri-miR-18a and also show reduced abundances of endogenous pre-miR-18a. Furthermore, we show that hnRNP A1 is required for miR-18a–mediated repression of a target reporter in vivo. These results underscore a previously uncharacterized role for general RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.

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Figure 1: Identification of a binding site for hnRNP A1 in pre-miR-18a.
Figure 2: hnRNP A1 binds pri-miR-18a but not other pri-miRNAs in the same cluster.
Figure 3: hnRNP A1 facilitates Drosha-mediated processing of miR-18a in vitro.
Figure 4: Chimeric constructs reveal the influence of context in pre-miR-18a production.
Figure 5: hnRNP A1 increases the abundance of endogenous processed miR-18a.

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Acknowledgements

We thank N. Hastie for comments and critical reading of the manuscript, and A. Krainer (Cold Spring Harbor Laboratory) for the generous gift of SF2/ASF antibody and recombinant hnRNP A1 protein. This work was supported by the Medical Research Council and Eurasnet (European Alternative Splicing Network-FP6). S.G. was supported by a European Molecular Biology Organization long-term postdoctoral fellowship.

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Authors and Affiliations

Authors

Contributions

S.G. and J.F.C. conceived, designed, and interpreted the experiments. S.G. performed the experiments and data analysis. J.F.C. supervised the whole project. The manuscript was cowritten by both authors.

Corresponding author

Correspondence to Javier F Cáceres.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Northern blotting of in vitro processing reactions assesses the identity and migration of individual precursor microRNAs. (PDF 2005 kb)

Supplementary Fig. 2

RNA interfererence–induced depletion of hnRNP A1 in HeLa cells. (PDF 272 kb)

Supplementary Fig. 3

Recombinant SF2/ASF cannot rescue the defect in in vitro miR-18a processing in extracts from cells depleted of hnRNP A1. (PDF 1318 kb)

Supplementary Fig. 4

Precursor miR-18a is not unstable in the absence of hnRNP A1. (PDF 2803 kb)

Supplementary Fig. 5

Alignment of precursor miR-18a and precursor miR-18b sequences reveals a close homology. (PDF 64 kb)

Supplementary Fig. 6

hnRNP A1 increases the abundance of endogenously processed miR-18a. (PDF 2205 kb)

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Guil, S., Cáceres, J. The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a. Nat Struct Mol Biol 14, 591–596 (2007). https://doi.org/10.1038/nsmb1250

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