The RNA Binding Protein RBM38 (RNPC1) Regulates Splicing during Late Erythroid Differentiation

Laurie A Heinicke,J Eric Russell,Behnam Nabet,Benjamin Cieply,Russ P Carstens,Sebastiaan Van Zalen,Peng Jiang,Yi Xing,Shihao Shen,Bin Tian

doi:10.1371/journal.pone.0078031

Laurie A Heinicke, J Eric Russell + Show 8 more

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https://doi.org/10.1371/journal.pone.0078031

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Abstract

Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71+ erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation.

Highlights

Alternative splicing of pre-mRNA transcripts generates protein isoforms with cell-type specific functions that are essential for development and homeostasis [1]
We present evidence supporting the role of RBM38 as a regulator of alternatively spliced transcripts during erythroid terminal differentiation
The inclusion of EPB41 exon 16, which produces a protein isoform that is necessary for the malleability of red blood cells, is the best characterized alternative splicing event during erythroid differentiation

Summary

Introduction

Alternative splicing of pre-mRNA transcripts generates protein isoforms with cell-type specific functions that are essential for development and homeostasis [1]. In addition to the ESRPs, this screen identified several other proteins that had not previously been shown to regulate splicing Among those regulators was RBM38 ( known as RNPC1), which robustly altered splicing of the reporter, but was not required for regulation of endogenous FGFR2 splicing [5]. RBM38 has not been characterized as an alternative splicing factor, but has been shown to regulate transcript stability by binding the 3’-UTR of cell cycle regulators p21, p53, p63 and p73[11,12,13,14,15,16,17,18], along with stabilizing mRNA transcripts of the multifunctional RNA-binding protein, HuR[14], and the murine double minute-2 (MDM2) oncogene [12]. Given the presumed role of RBM38 in alternative splicing, we set out to identify its endogenous targets

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