title: TIA proteins are necessary but not sufficient for the tissue-specific splicing of the myosin phosphatase targeting subunit 1.
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.
authors.affiliation: Departments of Medicine, Case Western Reserve University school of Medicine, Cleveland, Ohio 44106-4958, USA.
authors.forename: Supriya
authors.initials: S
authors.lastname: Shukla
authors.forename: Wessel P
authors.initials: WP
authors.lastname: Dirksen
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Joyce
authors.forename: Caroline
authors.initials: C
authors.lastname: Le Guiner-Blanvillain
authors.forename: Richard
authors.initials: R
authors.lastname: Breathnach
authors.forename: Steven A
authors.initials: SA
authors.lastname: Fisher
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D005091:Exons / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D005895:Gizzard, Avian / Q000166:cytology / Q000502:physiology; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000502:physiology*; D043263:Myosin-Light-Chain Phosphatase / Q000235:genetics* / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D022821:RNA Splice Sites / Q000502:physiology; D012326:RNA Splicing / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D011506:Proteins; D022821:RNA Splice Sites; D016601:RNA-Binding Proteins; D043263:Myosin-Light-Chain Phosphatase