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Rbfox3 controls the biogenesis of a subset of microRNAs

Nature Structural & Molecular Biology volume 21, pages 901–910 (2014)Cite this article

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Abstract

RNA-binding proteins (RBPs) regulate numerous aspects of gene expression; thus, identification of their endogenous targets is important for understanding their cellular functions. Here we identified transcriptome-wide targets of Rbfox3 in neuronally differentiated P19 cells and mouse brain by using photoactivatable ribonucleoside–enhanced cross-linking and immunoprecipitation (PAR-CLIP). Although Rbfox3 is known to regulate pre-mRNA splicing through binding the UGCAUG motif, PAR-CLIP analysis revealed diverse Rbfox3 targets including primary microRNAs (pri-miRNAs) that lack the UGCAUG motif. Induced expression and depletion of Rbfox3 led to changes in the expression levels of a subset of PAR-CLIP-detected miRNAs. In vitro analyses revealed that Rbfox3 functions as a positive and a negative regulator at the stage of pri-miRNA processing to precursor miRNA (pre-miRNA). Rbfox3 binds directly to pri-miRNAs and regulates the recruitment of the microprocessor complex to pri-miRNAs. Our study proposes a new function for Rbfox3 in miRNA biogenesis.

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Figure 1: PAR-CLIP for endogenous Rbfox3 in neuronally differentiated P19 cells.
Figure 2: Rbfox3-regulated miRNAs in P19 cells.
Figure 3: Effects of Rbfox3 on miRNA biogenesis in P19 cells.
Figure 4: Binding of Rbfox3 to pri-miRNAs in P19 cells and in vitro.
Figure 5: Differential RNA binding activities and pre-miRNA generating activities among Rbfox isoforms.
Figure 6: Determination of Rbfox3-binding regions in pri-miRNAs.
Figure 7: Direct effects of Rbfox3 on pri-miRNA processing into pre-miRNA.
Figure 8: Regulation of pri-miRNA processing by Rbfox3.

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Acknowledgements

We thank U. Ohler, D.L. Corcoran and N. Mukherjee (Duke University) for help in PARalyzer analysis, H. Wu (Lawrence Berkeley National Laboratory) for advice on preparation of a sequence library for PAR-CLIP, D.-Y. Lee (Biochemistry Core Facility, US National Heart, Lung, and Blood Institute (NHLBI)) for HPLC analysis of ribonucleosides and C.A. Combs (Light Microscope Core Facility, NHLBI) for advice on confocal microscopy. We also thank D.E. Saunders and A.F. Smith for technical assistance, S. Nakahata for reagents and M.A. Conti for critical reading of the manuscript. This work was supported by the Division of Intramural Research, NHLBI, US National Institutes of Health.

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Authors and Affiliations

  1. Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

    Kee K Kim, Robert S Adelstein & Sachiyo Kawamoto

  2. DNA Sequencing and Genomics Core, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

    Yanqin Yang & Jun Zhu

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Contributions

K.K.K. and S.K. designed the study. K.K.K. performed the biological experiments, and K.K.K., R.S.A. and S.K. interpreted the data. Y.Y. and J.Z. performed bioinformatics analyses. K.K.K., R.S.A. and S.K. wrote the manuscript with input from Y.Y. and J.Z.

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Correspondence to Kee K Kim or Sachiyo Kawamoto.

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Integrated supplementary information

Supplementary Figure 1 In vivo PAR-CLIP for Rbfox3 in the mouse CNS.

(a) Schematic outline of the strategy for in vivo mouse PAR-CLIP. Intraperitoneal injection of 4SU into adult mice and irradiation of dissociated cells from tissues with 365 nm UV followed by common PAR-CLIP protocol are illustrated. (b) Autoradiogram of radioactively phosphorylated RNA–protein complexes following LDS-PAGE. Samples are T4 polynucleotide kinase-phosphorylated anti-Rbfox3- or IgG-immunoprecipitates prepared from UV-irradiated brain cells of 4SU injected mice. The area boxed with broken red lines was processed for further analysis. IP, immunoprecipitation. (c) Immunoblot detecting Rbfox3. Samples are immunoprecipitates with mouse anti-Rbfox3 (m) or IgG from mouse brain extracts. Rabbit anti-Rbfox3 (r) was used for immunoblotting. Input contains 5% extract. Uncropped image is provided in Supplementary Figure 5. (d) Genomic distribution of Rbfox3 binding clusters. Numbers in parenthesis indicate cluster numbers. A full list of PAR-CLIP clusters is provided in Supplementary Data Set 2.

Supplementary Figure 2 Effect of Rbfox3 on pri-miRNA levels in Drosha-knockdown P19 cells.

Transfected siRNA or expression plasmid is indicated. siDrosha, siRNA against Drosha; control, non-targeting siRNA and the empty vector. (a) Quantification of pri-miRNAs by qRT-PCR. n = 3 (biological replicates, cell cultures), average ± s.e.m., * P < 0.001 (ANOVA, Bonferroni’s multiple comparisons test). ns, no statistically significant difference. (b) RT-PCR analysis of pri-miRNAs. PCR products with (+) or without (−) RT following agarose gel electrophoresis is shown. (c) Immunoblot analysis of Drosha and myc-Rbfox3. Gapdh serves as a loading control. Uncropped images are provided in Supplementary Figure 5.

Supplementary Figure 3 Nucleotide sequences of the stem-loop region of wild-type and mutant pri-miRNAs.

Mature miR-5p and 3p are indicated in red. Mutant nucleotides are indicated in cyan and dots indicate deletion. The underlined region corresponds to the Rbfox3-binding cluster detected by PAR-CLIP. Wt, wild-type; Mt, mutant.

Supplementary Figure 4 Determination of 4SU residues on pri-miRNAs cross-linked with Rbfox3 by in vitro PAR-CLIP.

The “u” residues in red were converted to “c”, indicating the crosslinked residues. (a,b) The nucleotides with underlines were the cluster sequences determined in RA-treated P19 cells. (c) The underlined sequences are the canonical Rbfox binding sequence, UGCAUG, in the IDDE.

Supplementary Figure 5 Uncropped images of immunoblots and autoradiograms.

The areas boxed with broken red lines are used for the figures.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 3670 kb)

Supplementary Data Set 1

Rbfox3-binding clusters in neuronally differentiated P19 cells determined by PAR-CLIP (XLSX 418 kb)

Supplementary Data Set 2

Rbfox3-binding clusters in the mouse brain and spinal cord determined by PAR-CLIP (XLSX 97 kb)

Supplementary Data Set 3

Comparative miRNA microarray analysis of untreated P19-GFP, RA-treated P19-GFP and RA-treated P19-T2 cells (XLSX 24 kb)

Supplementary Data Set 4

PAR-CLIP read numbers at each processing step from P19 cells and mouse brain (XLSX 9 kb)

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Kim, K., Yang, Y., Zhu, J. et al. Rbfox3 controls the biogenesis of a subset of microRNAs. Nat Struct Mol Biol 21, 901–910 (2014). https://doi.org/10.1038/nsmb.2892

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