Alternative of this AS event have yet to

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.

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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.