Crosstalk between PTBP1 and miR‐101/AGO2 on Targeting MCL1 – A Novel Post‐Transcriptional Mechanism for MCL1 Expression

Background Myeloid Cell Leukemia 1 (MCL1) is a key anti‐apoptotic protein belonging to the BCL‐2 protein family. To preserve normal cellular homeostasis, cells must maintain strict control over MCL1 expression. Overexpression of MCL1 has been identified as a key contributor to tumorigenesis and further enables resistance to a number of anti‐cancer chemotherapies. Thus, there is an ongoing interest to develop selective MCL1 inhibitors in anti‐cancer therapies. In order to better target MCL1, it is essential to understand the molecular mechanisms that regulate MCL1 expression in cells. Previous studies have detailed how MCL1 expression is controlled by multiple mechanisms transcriptionally, translationally, and post‐translationally. However, minimal characterization of the post‐transcriptional regulators of MCL1 mRNA is available. Polypyrimidine tract binding protein 1 (PTBP1) is a RNA binding protein that is known to regulate gene expression post‐transcriptionally via its control over mRNA processing and metabolism. Our previous work identified PTBP1 as a MCL1 repressor by binding to and destabilizing MCL1 mRNA. In the described study, we will further characterize the corresponding mechanisms of the PTBP1‐mediated MCL1 repression especially on the crosstalk between PTBP1 and microRNA (miRNA), and their roles on apoptosis and drug responses. Methods RNA immunoprecipitation (RIP) and cross‐linking immunoprecipitation sequencing (CLIP‐seq) data analysis were performed to characterize protein‐mRNA interactions and localize RNA binding sites on the genes of interests. siRNAs and miRNA mimics were transiently transfected into cancer cell lines using lipofectamine reagents. miRNA expression was quantified using TaqMan assays. Gene expression was quantified by western blotting for protein level and RT‐qPCR for mRNA level. Apoptosis was assessed by Annexin V/Propidium Iodide (PI) staining followed by flow cytometry analysis, and cell survival was measured by colony formation assay. Results Our studies demonstrate that the increase of MCL1 by PTBP1 knockdown is not simply due to a corresponding decrease in the production of the MCL1 ‐targeting miRNAs. Instead, PTBP1 silencing reduces Argonaute2 (AGO2) association with MCL1 . AGO2‐CLIP‐seq analysis further identifies the miR‐101 seed site on MCL1 3′ untranslated region (3′ UTR) with dramatically decreased AGO2 binding upon PTBP1 knockdown. Moreover, we show that PTBP1 knockdown inhibits miR‐101‐induced AGO2 binding to MCL1 and blocks miR‐101's repression on MCL1 expression. As a result, PTBP1 regulates miR‐101's function on apoptosis and cell survival. Conclusions Our findings suggest that PTBP1 modulates MCL1 expression by regulating miRNA‐direction of the miRNA induced silencing complex (miRISC) to MCL1 . We demonstrate that PTBP1 enhances miR‐101‐guided AGO2 interaction with MCL1 , thereby regulating miR‐101 induced apoptosis and clonogenic cell survival inhibition in cells. Support or Funding Information This work was supported by the National Institutes of Health [RO1 GM117391 to W.J.P.]; the UAB New Faculty Development Grant (to W.J.P.); the UAB Comprehensive Cancer Center New Faculty Development Award in Cancer Research (to W.J.P.); the UAB HHMI Med to Grad Fellowship (to J.C.); and the UAB Carmichael Scholarship (to J.C.). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .