Regulation of CD4 T cell and HIV-1 gene expression by Sam68

Humans have ~20,000 genes and over 50 trillion cells. However, the C. elegans worm has ~20,000 genes and ~1000 cells. Therefore, human complexity is not determined by the number of genes. Instead, human genes are highly regulated in terms of how many different proteins can be made from a single gene using processes such as alternative splicing and alternative polyadenylation. Also, genes are controlled in terms of when they are expressed by being transcribed into RNA and how efficiently this RNA is translated into protein. There is still little understanding of how RNA binding proteins control how many different proteins and how much of a specific protein is made from a gene. This proposal will determine how the cellular RNA binding protein Sam68 controls RNA abundance from specific genes, how efficiently this RNA is translated into protein, and how many different proteins can be made from that gene. We will analyse this in human CD4 T cells, which are an essential component of the immune system that protects the body from infections. We will determine how the location of Sam68 binding sites on an RNA controls its ability to regulate which proteins are expressed from a gene through alternative splicing. We will also analyse whether the genes that Sam68 regulates control important functions for the immune system. Sam68 is implicated in several types of cancer including breast, prostate, colorectal, cervical and renal cell carcinoma as well as the diseases fragile X-associated tremor/ataxia syndrome (FXTAS) and spinal muscular atrophy (SMA). Understanding how Sam68 controls gene expression may also help us understand how Sam68 regulates these diseases.

HIV infection causes AIDS and is a worldwide pandemic. Currently, there are approved antiviral drugs targeting four steps of the HIV life cycle. However, drug resistant strains of HIV are a problem and there is still a great need for new drug targets. One promising approach to develop novel antiviral drugs is to target cellular proteins. HIV has only nine genes and must highjack hundreds of cellular proteins to replicate. Sam68 regulates HIV gene expression but it is unclear how it does so. It is also unknown if HIV infection alters which cellular proteins are expressed by interacting with Sam68, especially if the cell and virus have to compete for it. Therefore, we will determine how HIV interacts with Sam68 to control cellular and viral gene expression. This may determine if Sam68 or other steps of HIV gene expression are potential drug targets.

Sam68 is a cellular RNA binding protein that controls multiple steps of cellular gene expression and also regulates HIV-1 gene expression. To determine how Sam68 controls gene expression in primary human CD4 T cells, we will use RNA-seq to determine which RNAs change their abundance, alternative splicing, poly(A) site selection and translation efficiency upon T cell activation in a Sam68-dependent manner. We will then identify in vivo Sam68 RNA binding sites and use Bayesian networks to develop a predictive Sam68 target network. We will also determine if Sam68 controls the expression of functionally related proteins or pathways. Overall, these experiments will identify 1) the specific human CD4 T cell mRNAs that Sam68 regulates on a genome-wide level, 2) whether these mRNAs encode proteins that control related immunological functions, 3) the specific step(s) of gene expression that Sam68 regulates and 4) how the location of Sam68 RNA binding sites controls alternative splicing. This information can be used to understand how Sam68-regulated cellular RNAs control activated CD4 T cell function.

We will also characterise how HIV-1 infection of primary human CD4 T cells affects Sam68-regulated cellular RNAs and how Sam68 regulates HIV-1 gene expression. This will allow two important analyses. First, we will determine if HIV-1 infection regulates cellular gene expression by altering Sam68 activity or availability, which could alter the expression of immunologically active proteins. Second, we will compare how Sam68 regulates cellular and HIV-1 RNAs. It will be interesting to determine if HIV-1 RNAs interact with Sam68 in an identical manner to cellular mRNAs, if a subset of cellular mRNAs are regulated more similarly to HIV-1 RNAs than other cellular mRNAs, or if HIV-1 RNAs interact with Sam68 differently than cellular mRNAs to control viral gene expression, which may indicate that Sam68 is a potential antiretroviral target.

The primary objective of this proposal is to determine how Sam68 controls CD4 T cell and HIV-1 gene expression. This research will have several different types of impact. First, the experiments in this grant will determine how Sam68 regulates CD4 T cell gene expression, which could provide insight into how CD4 T cells help control infection and are abnormally regulated in autoimmune diseases. Second, this proposal characterises how Sam68 regulates HIV-1 gene expression and replication, which could lead to new insights into HIV-1 post-transcriptional control mechanisms and novel antiretroviral treatments. It also characterises how HIV-1 infection may alter cellular gene expression by inhibiting the interaction between Sam68 and cellular RNAs. The development of new anti-HIV drugs remains a critical priority for combating the AIDS pandemic and targeting cellular factors is an exciting strategy for drug development. Maraviroc, which inhibits viral entry into the cell by binding CCR5, is the first approved antiretroviral that targets a host protein. Small molecules targeting other cellular RNA binding proteins that regulate HIV-1 replication, such as the SR protein SRSF1 and the RNA helicase DDX3, have been developed and inhibit HIV replication in vitro. This proposal may validate Sam68 as a potential antiretroviral drug target and, by increasing our understanding of HIV-1 gene expression, identify other novel antiretroviral drug targets. We will engage KCL's Business and Innovation department to evaluate the commercial potential of any discoveries that we make and, if warranted, help foster collaboration with pharmaceutical partners to develop novel therapeutics. Third, characterizing how Sam68 controls cellular gene expression at a genome wide level, and developing a predictive Sam68 target network, may allow the development of new insight and therapies into other diseases that Sam68 has been implicated in. These diseases include fragile X-associated tremor/ataxia syndrome (FXTAS), spinal muscular atrophy (SMA) and several types of cancer such as breast, prostate, colorectal, cervical and renal cell carcinoma. Fourth, this grant will employ two postdoctoral associates and they will receive training in cutting edge molecular biology and bioinformatics technology. The proposed RNA-seq and iCLIP experiments are at the forefront of molecular biology and the integration of the different types of data via the bioinformatics approaches will teach the postdocs how to answer complex biological questions that require an interdisciplinary team. They will also learn how to apply the molecular biology of how Sam68 regulates gene expression to virology and immunology scientific questions. These skills will be useful for a range of future career options including academic and pharmaceutical. Therefore, the beneficiaries of this research will potentially include: 1) scientists studying CD4 T cell gene expression and function, 2) scientists and companies studying HIV-1 gene expression and who are developing novel anti-HIV therapeutics, 3) scientists and companies who are studying FXTAS, SMA or several types of cancer in which Sam68 is upregulated as well as the patients who have these diseases and 4) the two postdoctoral associates who will receive further training in molecular biology, immunology, virology and bioinformatics.