Negative autoregulation of BMP dependent transcription by SIN3B splicing reveals a role for RBM39.

Noel Faherty,Daniel Ebner,Eshita Sharma,Matthew Benson,Alison Howarth,Angela Lee,Shoumo Bhattacharya,Helen Lockstone

doi:10.1038/srep28210

Noel Faherty, Daniel Ebner + Show 6 more

Open Access

https://doi.org/10.1038/srep28210

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Abstract

BMP signalling is negatively autoregulated by several genes including SMAD6, Noggin and Gremlin, and autoregulators are possible targets for enhancing BMP signalling in disorders such as fibrosis and pulmonary hypertension. To identify novel negative regulators of BMP signalling, we used siRNA screening in mouse C2C12 cells with a BMP-responsive luciferase reporter. Knockdown of several splicing factors increased BMP4-dependent transcription and target gene expression. Knockdown of RBM39 produced the greatest enhancement in BMP activity. Transcriptome-wide RNA sequencing identified a change in Sin3b exon usage after RBM39 knockdown. SIN3B targets histone deacetylases to chromatin to repress transcription. In mouse, Sin3b produces long and short isoforms, with the short isoform lacking the ability to recruit HDACs. BMP4 induced a shift in SIN3B expression to the long isoform, and this change in isoform ratio was prevented by RBM39 knockdown. Knockdown of long isoform SIN3B enhanced BMP4-dependent transcription, whereas knockdown of the short isoform did not. We propose that BMP4-dependent transcription is negatively autoregulated in part by SIN3B alternative splicing, and that RBM39 plays a role in this process.

Highlights

Members of the BMP/TGFβsuperfamily play critical roles in pluripotency, embryonic stem cell differentiation and developmental patterning[1]
We examined induction of Id1, Id2 and Smad[6] mRNA in response to BMP4 over a 24 hour period and found that knockdown of PABPN1, PHF5A, RBM39 or DHX15 resulted in increased expression of these target genes, which was detectable by 3/6 hours and sustained at 24 hours (Fig. 1D–F)
We identified nine targets involved in mRNA splicing and processing, representing a small subset of over 200 individual proteins in the spliceosome[24]

Summary

Introduction

Members of the BMP/TGFβsuperfamily play critical roles in pluripotency, embryonic stem cell differentiation and developmental patterning[1]. We used siRNA screening to identify novel negative regulators of BMP signalling. We identified a subset of RNA binding/mRNA splicing factors, including RBM39, which negatively regulate BMP4-dependent transcription. We propose that BMP4-dependent transcription is negatively autoregulated in part by SIN3B splicing, and that RBM39 plays a role in this process. Cells were transfected with ON-TARGETplus (OTP) SMARTpool or Silencer Select siRNAs for 24 hours followed by treatment with BMP4 for 24 hours, with the exception of a control treated with diluent only (Vehicle). C2C12 cells were transfected with OTP SMARTpool siRNAs for 24 hours followed by treatment with BMP4 for the hours indicated. Cells were transfected with siRNA for 24 hours, followed by treatment with BMP4 for 72 hours. Y-axis shows BMP4 induced alkaline phosphatase (mean +/−S.E.M., n = 3), normalised to total protein.

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