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Consistent with the role of microRNAs (miRNAs) in downregulating gene expression by reducing the translation and/or stability of target messenger RNAs1, the levels of specific miRNAs are important for correct embryonic development and have been linked to several forms of cancer2-4. However, the regulatory mechanisms by which primary miRNAs (pri-miRNAs) are processed first to precursor miRNAs (pre-miRNAs) and then to mature miRNAs by the multiprotein Drosha and Dicer complexes5-8, respectively, remain largely unknown. The KH-type splicing regulatory protein (KSRP, also known as KHSRP) interacts with single-strand AU-rich-element-containing mRNAs and is a key mediator of mRNA decay9,10. Here we show in mammalian cells that KSRP also serves as a component of both Drosha and Dicer complexes and regulates the biogenesis of a subset of miRNAs. KSRP binds with high affinity to the terminal loop of the target miRNA precursors and promotes their maturation. This mechanism is required for specific changes in target mRNA expression that affect specific biological programs, including proliferation, apoptosis and differentiation. These findings reveal an unexpected mechanism that links KSRP to the machinery regulating maturation of a cohort of miRNAs that, in addition to its role in promoting mRNA decay, independently serves to integrate specific regulatory programs of protein expression.
We analysed the immunopurified Dicer-containing complex11 by mass spectroscopy and identified, amongst other proteins, KSRP (Supplementary Fig. 1a)-a highly conserved nucleo-cytoplasmic RNA-binding protein regulating distinct steps of mRNA life cycle12,13 (Supplementary Fig. 1b-d). KH domains 3 and 4 of KSRP (KH3-4) are required to promote AU-rich element (ARE)-containing labile mRNA decay9,10. Coimmunoprecipitation revealed that KSRP is an integral component of the Dicer complex in HeLa cells (Fig. 1a). On Dicer-knockdown-induced (Supplementary Fig. 2a) pre-miRNA upregulation, an anti-KSRP antibody immunoprecipitated pre-let- 7a14 (Fig. 1b). Recombinant KSRP directly interacted with pre-let- 7a-1, and KH3-4 accounted for the high-affinity binding to RNA (Supplementary Fig. 3a). KSRP interacted with the terminal loop (TL) of pre-let-7a-1 (TL-let-7a-1) but did not associate with either single- or double-stranded mature let-7a (Fig. 1c), with KH3-4 accounting for KSRP binding to TL-let-7a-1 (Supplementary Fig. 3b).
We titrated the protein with increasing amounts of TL-let-7a-1, and TL-let-7a-1 with increasing amount of protein, while monitoring the binding by NMR and circular dichroism, respectively. KH3-4 binds to TL-let-7a-1 with a 1:1 stoichiometry and a...