Alternative gene splicing(AS) is one of the major contributors to proteome diversity, playing anessential role in normal development of humans. Contrastingly,aberrant AS is recognized as a driver of cancer, though thespecific factors and mechanisms that produce oncogenic splice variants arepoorly understood. One exampleof oncogenic AS is the AML1-ETO9a splice variant in acute myeloidleukemia (AML) patients with the t(8;21) chromosomal translocation. AML is caused by abnormal and rapid proliferationof myeloid progenitor cells. It is the most common cause of acute leukemia inadults.
In t(8;21) AML patients, thechimeric protein, AML1-ETO, is formed. This protein retains the DNA-bindingspecificity of AML1 and the ability to recruit ETO-associated co-repressors to supportself-renewal of hematopoietic progenitor cells. While AML1-ETO expression caninduce myeloproliferative disorders in mice, its expression alone is notsufficient for leukemogenesis. In contrast, asplice variant of AML1-ETO, including exon 9a, is sufficient to induce leukemogenesis. Regulators of this AS event have yet to beidentified. To identify these modulators, it is pivotal to employ a methodthat will target the pre-spliced messenger RNA (mRNA) of AML1-ETO along withthe upstream splicing factors. Recently, the clustered regularly interspaced shortpalindromic repeat (CRISPR) family member Cas9, was shown to targetspecific endogenous RNA molecules, with the help of a guide RNA (gRNA) sequenceand an additional oligonucleotide probe. Based on this finding, wepropose to develop a novel CRISPR-assisted proteomic method to identify RNAbinding proteins (RBPs) that interact with the AML1-ETO pre-mRNA in t(8;21) AMLcells.
Objectives: Aim 1: Prepare CRISPR components for AML-ETO pre-mRNA enrichment. CRISPR components were assembled as follows: dCas9 (a nuclease-dead Cas9 mutant) was expressed in Escherichia Coli BL21DE3 and purified with a HisTrap Column. dCas9 was then tagged with a biotinlabeling reagent (ThermoScientific). For gRNA, different sequences weredesigned to target AML1-ETO pre-mRNA and ordered as DNA oligos (Mobix). Oligoswere then transcribed using a transcription kit (NEB).
DNA probes for Cas9 targeting were designed to forma short DNA/RNA double stranded region upstream of the gRNA targets to enabledCas9 binding. Probes were ordered from Mobix. Aim 2: Determine RBPs associated withAML1-ETO pre-mRNA in t(8;21) AML cells.
Briefly,t(8;21) AML cells will be irradiated with UV light to cross-link RNA moleculeswith their RBPs. These cells will be lysed and incubated with the biotin-dCas9,gRNA, and DNA probes. The sample will then be incubated with immobilizedstreptavidin beads. The enriched RBPs will be released by RNase digestion ofthe AML-ETO pre-mRNA and followed with the subsequent mass spectrometryworkflow for protein identification. Aim 3: Validate the RNA-proteininteractions with AML-ETO pre-mRNA in t(8;21) AML cells.
Weplan to perform Aim 2 methods along with western blots to confirm theRNA-protein interactions discovered in Aim 2. We will further confirm theseinteractions reciprocally, by isolating the identified RBPs and detecting theAML1-ETO pre-mRNA with real-time PCR. Significance:This study will identify key AS regulators that direct expression of theleukemogenic isoform of AML1-ETO. This method could also be applied to othersplice isoforms. Thus providing a tool for specific endogenous RBPidentification, which has not been shown before.