Paul Cassar

From

Paul Cassar

Supervisor: William Stanford, Institute of Medical Science

WiP Seminar, 16 December 2008

The Role of the RING Finger Protein Makorin-1 in Embryonic Stem Cell Self-Renewal

The viability of pluripotent cells as a therapeutic source is reliant on the improved knowledge of the molecular events controlling their derivation and fate (self-renewal versus commitment). Accordingly, to identify novel regulators of ESC fate, we combined temporal expression microarray analysis on early committed ESCs with promoter occupancy studies. In this study, Makorin-1 (Mkrn1) was identified to be transcriptionally co-regulated with known regulators of ESC pluripotency. The function of Mkrn1 in ESC self-renewal is currently unknown however; its expression is dependent on the undifferentiated state of the ESC. To further investigate the role of Mkrn1 in ESC self-renewal we induced Mkrn1 knockdown with shRNA in stable ESC clones. The knockdown of Mkrn1 hastened differentiation and led to a concomitant decrease in Oct4 mRNA and protein levels when cultured in self-renewal conditions. Conversely, the enforced expression of Mkrn1 in ESCs hinders differentiation when cultured in differentiation conditions as evident by higher Oct4 mRNA and protein levels. The data indicate that Mkrn1 functions as a novel regulator of ESC self-renewal; however, its mechanism of action remains unknown. Mass spectrometry analysis of the Mkrn1 protein interaction network in undifferentiated ESCs revealed that Mkrn1 interacts with a number of known mRNA-binding proteins suggesting that Mkrn1 regulates ESC fate through a previously uncharacterized post-transcriptional complex.

Current work is focused on identifying the subset of Mkrn1-bound transcripts in ESCs through the use of ribonucleoprotein immunoprecipitation-sequencing (RIP-Seq) analysis. Our goal is to construct a Mkrn1 post-transcriptional to further our insight into the regulatory networks that control ESC fate decisions.

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