RNA Decay of LINE-1 Retrotransposons as a New Mechanism to Maintain B Cell Genome Integrity

PROJECT SUMMARY/ABSTRACT Nearly half of our genome is made of retrotransposon sequences. Long INterspersed Element-1 (LINE- 1 or L1) is the only known autonomously active human retrotransposon and has amplified to over 500,000 copies in both human and mouse genomes over evolutionary time. L1 insertions into the genomes of somatic cells can contribute to a wide range of pathologies by detrimentally altering gene expression. Mammalian cells have evolved many clever ways to protect their genomes against an onslaught of L1 retrotransposition. Somatic cells primarily rely on epigenetic modifications for L1 silencing, such as histone and DNA CpG methylationg. However, these mechanisms are not entirely foolproof. Occasionally, cells appear to show signs of “leaky” L1s that manage to escape silencing and therefore carry the potential to wreak havoc across the genome. We do not fully understand how these derepressed, transcribed L1s are regulated in cells. Given that L1 is a unique source of genome instability, I will investigate how L1 RNA activity is mitigated in B cells. My preliminary data from sequencing and RT-qPCR-based assays suggest that the RNA exosome, an 11 subunit RNA processing/degradation complex, plays a crucial role in targeting and degrading L1 RNA in B cells. To this end, I will investigate whether aberrantly expressed L1s in B cells are controlled at the level of RNA catabolism. In Aim 1, I will use an in vitro L1 retrotransposition assay to determine whether RNA exosome restrains L1 RNA from “jumping” into the genome. In Aim 2, I will use biochemical and molecular approaches to assess whether m6A-binding protein YTHDC1 acts as an adaptor to specifically recruit exosome to L1 RNA. In Aim 3, I will use Nanopore long read sequencing to determine L1 retrotransposition “hotspots” and then characterize the cellular consequences of these insertions by examining proliferation, apoptosis, migratory potential, and susceptibility to future translocations. Ultimately, I aim to unveil a new role of L1 RNA suppression in B cells by RNA exosome-mediated RNA decay and characterize the cellular consequences in the absence of such faithful homeostatic RNA decay. This leap in our understanding of L1-mediated genome instability may eventually lead to ways of addressing B cell diseases originating from aberrant translocations or recombination events. With the support of the Kirschstein- NRSA fellowship and the stimulating environment of Columbia University’s MD/PhD program, I will grow scientifically, medically, and professionally in ways that will allow me to achieve my career goal of becoming a physician-scientist studying RNA, immunology, and pathophysiology through the lens of patient care.