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.

SummaryWe recently found that hnRNP A1, a protein implicated in many aspects of RNA processing, acts as an auxiliary factor for the Drosha-mediated processing of a microRNA precursor, pri-miR-18a. Here, we provide the mechanism by which hnRNP A1 regulates this event. We show that hnRNP A1 binds to the loop of pri-miR-18a and induces a relaxation at the stem, creating a more favorable cleavage site for Drosha. We found that approximately 14% of all pri-miRNAs have highly conserved loops, which we predict act as landing pads for trans-acting factors influencing miRNA processing. In agreement, we show that 2′O-methyl oligonucleotides targeting conserved loops (LooptomiRs) abolish miRNA processing in vitro. Furthermore, we present evidence to support an essential role of conserved loops for pri-miRNA processing. Altogether, these data suggest the existence of auxiliary factors for the processing of specific miRNAs, revealing an additional level of complexity for the regulation of miRNA biogenesis.

hnRNP A1 is a nucleocytoplasmic shuttling protein that is involved in many aspects of mRNA metabolism. We have previously shown that activation of the p38 stress-signaling pathway in mammalian cells results in both hyperphosphorylation and cytoplasmic accumulation of hnRNP A1, affecting alternative splicing regulation in vivo. Here we show that the stress-induced cytoplasmic accumulation of hnRNP A1 occurs in discrete phase-dense particles, the cytoplasmic stress granules (SGs). Interestingly, mRNA-binding activity is required for both phosphorylation of hnRNP A1 and localization to SGs. We also show that these effects are mediated by the Mnk1/2 protein kinases that act downstream of p38. Finally, depletion of hnRNP A1 affects the recovery of cells from stress, suggesting a physiologically significant role for hnRNP A1 in the stress response. Our data are consistent with a model whereby hnRNP A1 recruitment to SGs involves Mnk1/2-dependent phosphorylation of mRNA-bound hnRNP A1.