TIA Proteins Are Necessary but Not Sufficient for the Tissue-specific Splicing of the Myosin Phosphatase Targeting Subunit 1

Supriya Shukla,Wessel P Dirksen,Katherine M Joyce,Caroline Le Guiner-Blanvillain,Richard Breathnach,Steven A Fisher

doi:10.1074/jbc.m314138200

Supriya Shukla, Wessel P Dirksen + Show 4 more

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https://doi.org/10.1074/jbc.m314138200

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Abstract

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.

Highlights

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity
We identified several cis-enhancer elements (Fig. 1B) in the vicinity of the alternative exon 5Ј-splice site, including a proximal U-rich intronic sequence, a 67-nucleotide intronic enhancer, and an exonic splicing enhancer, that are necessary for the splicing of the MYPT1 alt exon [11]
TIA-1 and T-cell inhibitor of apoptosis-related (TIAR) trans-Activate MYPT1 Central Exon Splicing—Transfection of the MYPT1 wild type minigene expression plasmid (Fig. 1A) into cultured gizzard SMCs resulted in 89 Ϯ 2% exon-included transcripts as we previously reported [11] (Fig. 2A)

Summary

Introduction

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. Reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.

Results

Conclusion