B-cells are a critical element of the adaptive immune response, and deficiencies in B-cell development lead to a variety of congenital immunodeficiency syndromes as well as age-related immune decline. Post- transcriptional gene regulation has recently been appreciated to play a major role in developmental pathways. Elements that control post-transcriptional gene regulation in trans include RNA-binding proteins(RBPs), microRNAs (miRNAs) and other non-coding RNA. Our recent work suggests that particular RNA binding proteins can exert powerful effects on hematopoietic development. However, the contribution of RNA binding proteins, as a class, to the development of B-lymphocytes has not been systematically assessed. The advent of CRISPR/Cas9 genome editing technologies presents an unprecedented opportunity to study the function of proteins at a genome-wide level. However, the utility of this technique in understanding physiological processes in primary cells has remained limited. Here, we present an innovative proposal, combining sophisticated analysis of RNA expression data with a novel, hematopoietic optimized CRISPR/Cas9 screening system, as a method to study the effects of hundreds of genes on B-lymphocyte development. By using these methods, we will test our central hypothesis that RBPs, as a class, regulate B-cell development. Our preliminary data, derived from RNA-sequencing of B-cell subsets, shows that RBPs are dynamically regulated across development. In this proposal, we will employ novel bioinformatics analyses to describe developmental trajectories of RBPs and compare them with known regulatory genes in B-cell development. Furthermore, we propose to screen developmentally regulated RBPs for a biological function in B-cell development by performing CRISPR/Cas9-based forward genetic screens using our newly optimized retroviral vector system and in vitro B-cell culture system. By identifying RBP knockout alleles generated during the screen, we will identify RBPs that are required for the development of B-cells. By comparing bioinformatically predicted and functionally validated RBPs, we will characterize the utility of bioinformatics algorithms in predicting RBP function in development. In summary, this exploratory proposal represents the first characterization of this important class of post-transcriptional regulators as elements in the control of B-cell development.
Defects in B-cell development are associated with a range of human diseases, including congenital immunodeficiency states, age-related immune senescence, and primary bone marrow failure syndromes. Here, we will define how a class of proteins, namely those that bind to RNA, contribute to the development of B-cells. Our studies will provide the necessary biological framework for future targeting of novel pathways to facilitate the development of normal B-cells and thereby promote immune function in various disease states.
