A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA

Abstract

CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of “regulated instability” of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3′ untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed Δ1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the Δ1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally, using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.

Separate cis-trans Pathways Post-transcriptionally Regulate Murine CD154 (CD40 Ligand) Expression

“…hnRNP L Binds CD154 mRNA through the 3Ј-UTR CARE to Regulate Poly(A) Tail Length-The in vitro binding of PTB proteins to the human CD154 3Ј-UTR was previously shown (10,14,15). Similar approaches have shown that the RNA-binding protein hnRNP L exhibits remarkable binding specificity and affinity for CA repeats (18 -20).…”

“…330-nt region containing CURE and CARE (14,15). The cisacting instability element in this region was never precisely identified.…”

“…Studies of the increased CD154 mRNA stability seen in activated (24 h) human T cells as well as the D1.1 mutant of the Jurkat cell line demonstrated that PTB proteins from D1.1 cell cytoplasm interacted at three separate sites within this region (11,15). Deletions that eliminated PTB binding to the CD154 3Ј-UTR decreased the stability of CD154 mRNA seen in D1.1 cells.…”

Knockdown of polypyrimidine tract-binding protein suppresses ovarian tumor cell growth and invasiveness in vitro

“…It is now widely believed that PTB functions as a negative regulator of pre-mRNA splicing, blocking the inclusion of numerous alternative exons into mRNA (Black, 2003), including its own exon . Besides roles in splicing, PTB has also been implicated in the regulation of other aspects of RNA metabolism, such as premRNA polyadenylation (Castelo-Branco et al, 2004), mRNA stability (Kosinski et al, 2003;Knoch et al, 2004), mRNA export from the nucleus (Zang et al, 2001) and mRNA localization in the cytoplasm (Cote et al, 1999). In addition, PTB is involved in the control of cap-independent translation driven by the internal ribosomal entry site (IRES).…”

CD40 ligand gene and Kawasaki disease

“…(Table 1). Three SNPs in exon 5, SNP14, 15 and 140, were in close proximity to the previously described CU-rich element, 22,23 where members of the polypyrimidine tractbinding proteins (PTB) family bind and play a role in regulating CD40L expression at the level of post-transcriptional mRNA decay (Figure 1). The possible existence of rare deleterious mutations or polymorphisms altering translation of the protein has been ruled out by resequencing all five exons of CD40L in DNA derived from 81 probands of sibling cases.…”