PTBP proteins in T cell activation: Cellular and molecular mechanisms of action

The adaptive immune system, which is comprised of cells called T- and B- lymphocytes, has evolved to provide protection against harmful microorganisms and parasites. These cells provide long-lasting memory of previous infections and harnessing their function is key for effective vaccination. T cells are not all the same and can divided into groups, or subsets, which have specialized functions. These include CD4+ T cells called T follicular helper cells (TFH) which help B cells make antibodies and CD8+ T cells that kill cells infected with viruses or other germs.

The importance of T cells has prompted many investigators to identify the genes that control their development, survival and function. One major class of genes that control T cell fate and function encode proteins that work at the level of gene transcription, the process by which genes are converted into messenger RNA. The importance of this layer of control is universally accepted and is exemplified by numerous studies using mouse genetics that identify the key genes involved. However, an additional layer of control is also important; this is called post-transcriptional regulation of gene expression and it can regulate how long messenger RNA sticks around for and the tempo at which the messenger RNA converts its message into protein.

When compared to transcriptional regulation, little is known about how post-transcriptional regulation controls the development and function of T cells. We have discovered that a class of genes encoding RNA binding proteins carry out important post-transcriptional roles in T cells. What is unknown is how they affect the biology of T cells. Our proposed research is based on unpublished data that identifies key RNA binding proteins (RBP) necessary for the proper development and function of T cells.

To develop this research area we wish to understand, at the level of the whole organism, the redundant role of the RBPs in the function of T cells. We will study the phenotype of mutant mice using conditional gene targeting in T cells. These conditional systems will test for roles in development and maintenance of activated T cells. We will identify the directly bound targets of the RBP in both mouse T cells using assays of protein RNA interaction. We will investigate the mode of regulation of the targets.

The research is aimed at understanding a new mechanism that is necessary for T cell function and thus has implications for vaccine success and for autoimmunity-when T cells reacts against our own tissues. Importantly, we wish to understand how this novel mechanism interfaces with the mechanisms that we know most about -signaling and transcription.

This work is the key step towards elucidating a novel molecular mechanism of gene regulation in T cells. Appreciation of the importance and understanding the details of this mechanism has the potential to open up new possibilities for immunomodulation.

This proposal aims to understand the cellular and molecular mechanisms regulated in CD4+ and CD8+ T cells by the PTBP family of RNA binding proteins. Our objectives tie together to test a hypothesis that the PTBPs are direct downstream targets of tyrosine kinases in T cells and thus are a means for tyrosine kinase linked receptors on T cells (such as the TCR or cytokine receptors) to bring about changes in splicing or polyadenylation, mRNA decay or translation.

With BBSRC funding we have generated conditional mutants that allow deletion or expression of the PTBPs. To delete specifically in T cells we will use Cre based systems that have been thoroughly tested and allow tissue specific and temporally controlled gene deletion. These conditional systems will test for roles in T cell activation and in the maintenance of effector and memory T cells using models of immune challenge including influenza virus. We will also use antigen specific T cell receptor transgenes and adoptive transfer and mixed bone marrow chimaeras as these are powerful approaches to testing T cell functions in vivo.

To understand the mechanism of gene regulation by the RBP we will make use of a variety of methods to examine the transcriptome of mutant T cells. These include application of RNAseq to measure RNA abundance, splicing and polyadenylation; the use of ribosome profiling to identify effects at the level of translation; and the identification of the direct targets of the PTBPs using individual-nucleotide resolution Cross-Linking and immuno-precipitation (iCLIP). Integration of these orthogonal datasets is a powerful way to discover how pathways are regulated, and integrated with each other, by PTBPs.

We will study the phosphorylation of the PTBPs and their regulation by signaling pathways. We anticipate that we can link the abundance, localization or activity of the PTBPs to receptor systems on T cells that drive activation, survival and acquisition of effector function.

This proposal addresses the fundamental question relevant to all developmental biology systems of how genome-encoded information is interpreted. Transcription regulation is part of the answer to this, but the regulation of RNA at the post-transcriptional level has emerged as a deterministic factor also in many systems. Our work is an important step in generating an integrated understanding in the context of immune cells, but the principles we discover could be applicable to other developmental systems as well.

This is a novel research area with the opportunity for training with in vivo skills and state of the art molecular biology and bioinformatics approaches. Our training puts great emphasis on the postdocs performing both wet lab and bioinformatics analysis, including training in writing scripts, finding and using the data generated by others that is relevant to the project and providing input in what it takes to make a useful pipeline for analysis.

For the recognized researcher this is key opportunity to publish high quality papers that inform the research direction of others and by demonstrating productivity making the transition into a principal investigator position.