Roles of RNA-binding Proteins in Programming Gene Expression in Mammals

National Institute Of Allergy And Infectious Diseases

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Abstract

A major aim of the Integrative Immunobiology Section is to decipher gene expression programs that direct cell fates in the hematopoietic and immune systems, since perturbations to their genetic program underlie many human diseases such as cancer, immunodeficiency, autoimmunity, allergy and hematological diseases. We seek knowledge that will provide insights for understanding how a cell decodes the instructions programmed by its own genome in order to develop and function. In 2012, we discovered that the RNA-binding protein Lin28b is specifically expressed in pre-natal hematopoietic stem/progenitor cells (HSPCs) but not in adult counterparts from the bone marrow (Science 335: 1195-1200). Furthermore, this single RNA-binding protein can reprogram adult bone marrow HSPCs to attain fetal-like properties. In 2019, we reported that a second RNA-binding protein called Igf2bp3 can cooperate with Lin28b in engineering fetal-like HSPCs (Genes & Development 33: 1048-1068). This finding has implications for hematopoietic stem cell (HSC) transplantation in the clinic. The standard practice of using HSCs from an adult donor does not regenerate the immune system in layers or waves which normally occur during natural development. As a result, patients who receive HSC transplantation may not regenerate certain cell types that develop early in life such as subsets of innate-like lymphocytes such as natural killer T (NKT), B-1 and marginal zone B cell. Our work on Lin28b and Igf2bp3 demonstrated that in principle, it is possible to engineer mouse fetal-like HSCs. Currently, we are attempting to demonstrate the same can be accomplished with human HSCs. Such cells could be used for in utero HSC transplantation which could be used to cure sickle cell disease and beta-thalassemia as well as primary immunodeficiencies that can be diagnosed prior to birth. In addition, we hypothesized that Lin28b may be involved in the regulation of the switch from fetal to adult hemoglobin gene expression by erythroid progenitors that occurs around birth. In collaboration with Jeff Miller's lab (NIDDK), we determined that ectopic expression of LIN28B in CD34+ adult HSPCs resulted in expression of fetal hemoglobin upon erythroid differentiation in a primary cell culture system (Blood 122: 1034-41). Mechanistically, our finding is remarkable since this switch in globin gene expression during ontogeny is still not completely understood in molecular terms and now we have uncovered a novel mode of controlling this medically important switch. Most studies have concentrated on transcriptional regulation of the globin gene cluster; however, we find that the RNA-binding protein, LIN28B plays a major role in fetal hemoglobin gene (HBG1 and HBG2) expression in part by inhibiting the biogenesis of the LET-7 family of microRNAs. Importantly, we believe that our findings provide a novel avenue for treating sickle cell disease and beta-thalassemia. Despite decades of intense research, we do not have a cure for these two devastating diseases that affect numerous patients including children, and is a growing public health issue in Africa and Asia. It has been postulated that reactivation of fetal hemoglobin expression could cure beta-globinopathies, and LIN28B and/or IGF2BP3 may be target(s) for mediating this switch. Recently, we initiated an investigation in the RNA-binding protein called MATRIN3 (MATR3). This gene has been found to be mutated in familial amyotrophic lateral sclerosis (ALS); however, the mutations map to unannotated domains of the protein and thus it is not understood what aspect of MATR3 function is perturbed and how that results in this fatal neurodegenerative disease. Since, MATR3 is reported to bind to RNA, we performed Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) to identify its direct targets in a human cell line. Analysis of the PAR-CLIP data reveal that MATR3 binds primarily to introns of thousands of transcripts. In parallel, we have used degron and CRISPR technologies to ablate MATR3 expression in human cell lines. RNA-seq analysis and gene set enrichment analysis revealed that autoinflammation via the cGAS-STING pathway may be an unappreciated mechanism in pathogenesis of ALS. Finally, integrating the RNA-seq and PAR-CLIP datasets revealed that at least one direct target of MATR3 might undergo aberrant alternative splicing that has not yet been reported and may explain the observed autoinflammatory phenotype.