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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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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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.
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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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DOI: https://doi.org/10.1038/nsmb1250
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