Rbfox proteins regulate microRNA biogenesis by sequence-specific binding to their precursors and target downstream Dicer

Abstract

Rbfox proteins regulate tissue-specific splicing by targeting a conserved GCAUG sequence within pre-mRNAs. We report here that sequence-specific binding of the conserved Rbfox RRM to miRNA precursors containing the same sequence motif in their terminal loops, including miR-20b and miR-107, suppresses their nuclear processing. The structure of the complex between precursor miR-20b and Rbfox RRM shows the molecular basis for recognition, and reveals changes in the stem-loop upon protein binding. In mammalian cells, Rbfox2 downregulates mature miR-20b and miR-107 levels and increases the expression of their downstream targets PTEN and Dicer, respectively, suggesting that Rbfox2 indirectly regulates many more cellular miRNAs. Thus, some of the widespread cellular functions of Rbfox2 protein are attributable to regulation of miRNA biogenesis, and might include the mis-regulation of miR-20b and miR-107 in cancer and neurodegeneration.

Figure 1.

Rbfox proteins specifically bind to miR-20b and -107 precursor hairpins and repress their processing in vitro. (A) Electrophoretic mobility shift assay analysis of the Rbfox RRM binding to pre-miR-20b and -107. Truncated constructs of pre-miR-20b and -107 were used in these figures but the same results were obtained with the complete pre-miRNA stem-loops (Supplementary Figure S1C and D). (B) In vitro Drosha processing of pri-miR-20b and -107 in HeLa nuclear extract (NE) is inhibited by the Rbfox RRM at the indicated protein concentrations. Pri-miRNAs are uniformly radiolabeled. (C) In vitro Drosha processing of pri-miR-20b and -107 in HeLa nuclear extract is inhibited by full-length Rbfox1 and Rbfox2 proteins, but not by the Rbfox2 F/A mutant that abolishes binding to RNA. Quantification of the gel bands for pre-miRNAs in Drosha processing assays was carried out using ImagineJ and normalized to NE only.

Figure 2.

Conformational changes of pre-miR-20b and -107 stem loops upon Rbfox-RRM binding. SHAPE reactivity traces for pre-miR-20b (A) and pre-miR-107 (B) in free and complex forms (protein/RNA ratio is 1:1); reactivity is normalized to untreated samples. Secondary structures of pre-miR-20b (C) and -107 (D) in their free and Rbfox RRM-bound forms calculated using MC-Fold and MC-Sym by incorporating information provided by SHAPE and NMR analyses (35). Nucleotides are colored according to their normalized SHAPE reactivity with no data as gray, low reactivity (<0.4) as black, medium reactivity (0.4–0.85) as orange and high reactivity (>0.85) as red. Canonical and non-canonical base pairs are shown by bold lines with black dots and non-bold lines, respectively.

Figure 3.

Structural basis for recognition of pre-miR-20b by the Rbfox RRM: (A) superposition of NMR structures of free pre-miR-20b; (B) superposition of NMR structures pre-miR-20b (orange) in complex with Rbfox RRM (green); comparison of loop conformations in free (C) and complex (D) forms; structural models of the free full-length pre-miR-20b (E) and the complex of full-length pre-miR-20b with Rbfox RRM (109–208) (F) showing how the highly conserved C-terminal tail of the RRM can reach down into the stem-loop to provide additional contacts that are not possible with single-stranded RNA. Nucleotides are colored by their normalized SHAPE reactivity.

Figure 4.

Rbfox2 downregulates mature miR-20b and upregulates miR-20b downstream targets (A) Quantitative RT-PCR of mature miR-20b in SHSY5Y cells. Cells were collected 48 h after siRNA transfection with anti-Rbfox2 siRNA. CTRL: cells transfected with scrambled siRNA. n = 3 (biological replicates, cell cultures), average ± s.e.m., **P < 0.01, *P < 0.05 (one way ANOVA statistical test). (B) Immunofluorescence micrographs showing nuclear localization of FLAG-Rbfox2 expressed in MCF7 cells. Exogenously expressed FLAG-Rbfox2 was detected 48 h after transfection by immunostaining as indicated. Nuclei are stained by DAPI. (C) qRT-PCR of mature miR-20b in MCF7 cells. CTRL: empty vector; miR-20b: cells collected 24 h after transfection of vector containing pri-miR-20b; miR-20b + FLAG-Rbfox2: Day 0—cells were seeded; Day 1—FLAG-Rbfox2 plasmid transfection; Day 2—pri-miR-20b vector transfection (into the same well transfected at day 1 with FLAG-Rbfox2); Day 3—cell harvest. n = 3 (biological replicates, cell cultures), average ± s.e.m., **P < 0.01, ***P < 0.001 (one way ANOVA statistical test). (D) Immunoblot analysis of endogenous PTEN protein in MCF7 cells. CTRL: empty vector; miR-20b 24 and 48 h: cells collected 24 or 48 h after transfection of vector containing pri-miR-20b; miR-20b + Rbfox2: Day 0—cells were seeded; Day 1—FLAG-Rbfox2 plasmid transfection; Day2—pri-miR-20b plasmid transfection (into the same well transfected at day 1 with FLAG-Rbfox2); Day 3—cells harvested. α-actinin serves as a loading control. The levels of PTEN were quantified by densitometry and the normalized level in the presence of control vector was set to 1.

Figure 5.

Effects of Rbfox2 on the levels of mature miR-107 and its downstream target Dicer in mda-mb-231 cells. (A) qRT-PCR of mature miR-107. Transfected siRNAs are as indicated. Rbfox2 siRNA: siRNA against Rbfox2; CTRL siRNA: non-targeting siRNA. n = 3 (biological replicates, cell cultures), average ± s.e.m., *P < 0.05 (one way ANOVA statistical test). (B) Immunoblot analysis of Dicer and Rbfox2 proteins. Rbfox2–1A and Rbfox2–1F represent two different isoforms of this protein (75). Actinin serves as a loading control.