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.

CD154 (CD40L) mRNA turnover is regulated in part at the posttranscriptional level by a protein complex (termed Complex I) that binds to a highly CU-rich region of the 3′UTR. Polypyrimidine tract-binding protein (PTB) has previously been identified as a major RNA-binding protein in Complex I. Nondenaturing gel filtration of total extract from Jurkat T cells demonstrated that the CD154 mRNA-binding activity migrates as a ∼200-kDa complex, indicating the presence of multiple complex-associated proteins. We have currently undertaken a biochemical approach to further characterize Complex I and observed that it segregates over DEAE-Sepharose into two subcomplexes (termed I-L and I-U). Furthermore, nucleolin was identified as a component of both subcomplexes and was shown that it is the major RNA-binding protein in I-U. To directly demonstrate the biological significance of Complex I binding to the CD154 transcript, cytoplasm from human Jurkat cells was fractionated over a sucrose gradient and the different cellular fractions subjected to immunoprecipitation with anti-PTB and anti-nucleolin Abs. RT-PCR of the immunoprecipitated products using CD154-specific primers clearly demonstrated that nucleolin and PTB are associated with CD154 mRNA in both the ribonucleoprotein and polysome fractions. These data strongly support a model whereby nucleolin and PTB are integral to the stability of CD154 mRNA and are components of the CD154 ribonucleoprotein particle associated with actively translating ribosomes.

After several years of REDD+ (reducing emissions from deforestation and forest degradation in developing countries, and the role of conservation, sustainable management of forests, and enhancement of forest carbon stocks in developing countries) readiness, countries are starting to move toward REDD+ implementation and accessing results-based payments (RBPs). Currently various parallel processes for accessing RBPs exist, including project and jurisdictional—approaches that often operate under a nascent national framework. This review is structured around the key considerations for countries to implement REDD+ and access RBPs. It offers a discussion focusing on three areas that are crucial for the success of REDD+: ( a) REDD+ in the context of Nationally Determined Contributions (NDCs) under the Paris Agreement and the UN Sustainable Development Goals (SDGs), ( b) the role of the private sector in achieving emissions reductions, and ( c) access to RBPs for REDD+. We present some key considerations for future issues and possible successes of REDD+ implementation.

SummaryWe previously identified a cis-acting element within the 3 0 untranslated region of CD40 ligand messenger RNA (mRNA) that is composed of three complex binding sites and acts to increase mRNA stability in both in vitro and in vivo systems. We now demonstrate the functional consequences of the three binding sites with respect to increasing both luciferase activity and mRNA stability in a heterologous transcript expressed in a T-cell line. The internal region B was shown to be a bona fide stability element because its presence increased luciferase activity fourfold over the unmodified transcript and its removal from the XbaI-HaeIII region resulted in rapid degradation of the transcript. Region A contained both a binding site for a polypyrimidine-tract-binding protein (PTB)-mediated complex (Complex I) and an upstream, adjacent sequence that was a negative regulator of mRNA stability. Region C bound Complex II, which contained both PTB and heterogeneous nuclear ribonucleoproteinL (hnRNPL), and was less effective as a stability element on its own compared to region B. Our findings demonstrate differential levels of activity for the three binding sites relative to the turnover of CD40 ligand mRNA, suggesting that the lack of binding of Complex I/II during the early stages of T-cell activation contributes to the rapid degradation of the CD40 ligand mRNA transcript.

The expression of CD40L is tightly regulated at the posttranscriptional level throughout a time course of T cell activation, which corresponds to a significant increase in message stability at late times of activation (24-48 hr). Previous work revealed a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex binds to the CD40L 3’UTR at late times of T cell activation. We used RNA interference to downregulate PTB in D1.1 Jurkat T cell line to address the functional roles of PTB in CD154 mRNA stability and surface expression. Real-time qPCR measurement of the CD154 mRNA decay revealed that downregulating PTB caused a > 2-fold decrease in the CD40L mRNA half-life. The downregulation of PTB also caused an approximate 2-fold decrease in the mean fluorescence (MFI) of CD40L. Analysis of PTB cellular distribution during a time course of CD4+ T cell activation revealed cytoplasmic and nuclear localization in all resting and activated cells. However, there was an increase in expression at late times of activation. Binding studies revealed that CD40L mRNA is bound by nuclear PTB at all times of activation indicating that the requirements for binding of CD40L message by nuclear versus cytoplasmic PTB is highly distinct. Finally, the binding of CD40L message corresponded to a change in phosphorylation status of cytoplasmic PTB. These findings support a model whereby activation-induced phosphorylation of PTB is required for cytoplasmic binding to CD40L mRNA.