The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs

Abstract

Consistent with the role of microRNAs (miRNAs) in down-regulating gene expression by reducing the translation and/or stability of target messenger RNAs, the levels of specific miRNAs are important for correct embryonic development and have been linked to several forms of cancer. 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 complexes, 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 decay. 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.

Figure 1. KSRP, a component of Dicer complex, interacts with the TL of pre-let-7a-1

a, Coimmunoprecipitation of endogenous KSRP and either Dicer or Flag-TRBP in HeLa cell extracts. b, Coimmunoprecipitation of KSRP and pre-let-7a in HeLa cells transiently transfected with siDicer. c, KSRP (300 nM) binds to TL-let-7a-1 but not to single-strand (ss) or double-strand (ds) let-7a. d, Superimposition of ¹⁵N-¹H HSQC spectra of KH3-4 free (violet) and bound to TL-let-7a-1 (yellow). In the blow-up, spectra of bound KH3 (cyan) and KH2-3 (red). Arrows highlight the shift of a few selected peaks in the core of the RNA-binding groove. e, Interaction of KSRP (50-300 nM) with either wild-type (WT) TL-let-7a-1 or two distinct mutants.

Figure 2. KSRP regulates pre-let-7a-1 processing and controls the expression of select miRNAs

a, KSRP knock-down reduces the effect of endogenous let-7a on the activity of pGL3-let-7a6XBS. Student's t-test: * p-value < 0.05, ** p-value < 0.01. b, KSRP knock-down reduces the effect of either prilet-7a-1 or pre-let-7a-1, but not of mature let-7a, on pGL3-let-7a6XBS activity. * p-value < 0.05. c, recombinant KSRP increases the processing activity of recombinant Dicer on pre-let-7a. d, Analysis of total RNA from either control (siCtrl), KSRP, or Dicer knock-down HeLa cells. e, f, KSRP knock-down does not reduce the effect of a chimaeric pre-let-7a-1 comprising the TL of miR-23b on the activity of pGL3-let-7a6XBS (e) while impairing the effect of a chimaeric pre-miR-23b comprising the TL of pre-let-7a-1 on the activity of pGL3-miR-23b3XBS (f). * p-value < 0.05. All data are presented as mean ± s.d. (n=4).

Figure 3. KSRP is a component of the Microprocessor complex, interacts with pri-let-7a-1 favouring its processing, and is required for both Drosha and Dicer complexes interaction with let-7a precursors

a, Coimmunoprecipitation of endogenous KSRP with either Flag-Drosha or Flag-DGCR8. b, anti-KSRP immunoprecipitates select pri-miRNAs. c, KSRP (50 nM) restores pri-let-7a-1 processing when added to extracts from shKSRP stably transfected cells while KSRP overexpression enhances pri-let-7a-1 processing. d, e, KSRP knock-down impairs the interaction of Drosha and Dicer with select miRNA precursors. ** p-value < 0.01. All data are presented as mean ± s.d. (n=4).

Figure 4. KSRP affects let-7-regulated cell proliferation and is involved in Lin28-regulated maturation of let-7g in P19 cells

a, KSRP knock-down increases proliferation while inhibits the antiproliferative effect of transfected prilet-7a-1 but not of mature let-7a in U2OS cells. * p-value < 0.05, ** p-value < 0.01. b, KSRP interacts with pri-let-7g only in differentiated P19 cells. c, Lin28 knock-down in undifferentiated P19 cells induces the expression of let-7g. Concomitant Lin28 and KSRP knock-down abolishes let-7g expression. f, A model for KSRP-dependent regulation of processing of select miRNA. All data are presented as mean ± s.d. (n=4).