Polypyrimidine Tract-Binding Protein Is Critical for the Turnover and Subcellular Distribution of CD40 Ligand mRNA in CD4+ T Cells

Abstract

CD40 ligand (CD40L or CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable transcript at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-containing complexes (Complex I and II) that bind to three adjacent CU-rich sequences within the 3′ untranslated region (3′UTR). To assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using shRNA in both primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and the translating polysomes. The activation-induced formation of PTB-specific RNP complexes was observed only with cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that distinctly modified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA stability at late times of activation and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes thus leading to an enhanced level of CD40L protein.

PTBP1 modulation of MCL1 expression regulates cellular apoptosis induced by antitubulin chemotherapeutics

“…41 Similar studies have demonstrated PTBP1's ability to regulate subcellular distribution of STX1B, VAMP2, SV2A, KIF5 and CD40L mRNA. 29,42 To determine whether PTBP1 influenced MCL1 mRNA cellular distribution, we performed nucleo-cytoplasmic fractionation of both protein and RNA in siControl and siPTBP1 cells. Both protein lysates and RNA extracts from whole-cell, cytoplasmic and nuclear fractions were collected.…”

“…PTBP1 is a RNA binding protein that regulates RNA splicing, IRES (internal ribosome entry segment)-mediated translation initiation, 3′-end processing, mRNA turnover, localization, and transportation. 28 PTBP1 has been shown to be involved in distinct cellular processes including T-cell activation, 29 HCV replication and infection, 30 insulin secretion, 31 and apoptosis [32][33][34] through its post-transcriptional control of RNA. Recent studies have highlighted that PTBP1 expression is often dysregulated in human cancer, though its effect on malignancy appears to be cell-type dependent.…”

Neuronal regulation of pre-mRNA splicing by polypyrimidine tract binding proteins, PTBP1 and PTBP2

“…There are other phosphorylation sites on PTB in addition to Ser16, and other posttranslational modifications are also likely present. Cytoplasmic relocalization is presumably required for PTB’s known cytoplasmic functions such as the control of mRNA localization, translation, and mRNA stability (Hamilton et al, 2003; de Hoog et al, 2004; Fred et al, 2006; Lewis et al, 2008; Sawicka et al, 2008; Matus-Nicodemos et al, 2011). …”

“…These proteins, including the SR proteins, many of the original hnRNP proteins (notably A1, H, L), the CELF, Nova, Rbfox, and Muscle blind families, and others, belong to a variety of structural families and have different RNA recognition properties and different patterns of expression in tissues (Graveley, 2000; Dreyfuss et al, 2002; Black, 2003; Chen and Manley, 2009; Kalsotra and Cooper, 2011). In addition to their nuclear role in RNA processing, these proteins, including PTB, also often affect cytoplasmic processes such as mRNA localization, translation or decay (Hellen et al, 1993; Cote et al, 1999; Kim et al, 2000; Tillmar and Welsh, 2002; Hamilton et al, 2003; de Hoog et al, 2004; Bushell et al, 2006; Fred et al, 2006; Sawicka et al, 2008; Babic et al, 2009; Besse et al, 2009; Kafasla et al, 2009; Cobbold et al, 2010; Matus-Nicodemos et al, 2011). Within the nucleus, these proteins affect large programs of alternative processing events (Ule et al, 2006; Boutz et al, 2007b; Kalsotra et al, 2008; Licatalosi et al, 2008; Xue et al, 2009; Yeo et al, 2009; Kalsotra and Cooper, 2011).…”

CD5 expression is regulated during human T‐cell activation by alternative polyadenylation, PTBP1, and miR‐204

“…PTBP1 is a multifunctional protein, with well‐described functions in splicing, translation, polyadenylation, and mRNA stability. In T cells, PTBP1 has been shown to bind to the 3′‐UTR of the CD40 ligand ( CD154 ) mRNA, stabilizing it during activation of T cells, which corresponds to the temporal expression of CD154 . In other cell types, PTBP1 was shown to bind to cis ‐elements in the pre‐mRNA and modulate PAS usage .…”