טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGermanguz Igal
SubjectCharacterization of Adenosine to Inosine RNA Editing in
Human Embryonic Stem Cells and Reprogramming
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Joseph Itskovitz
Full Thesis textFull thesis text - English Version


Abstract

Adenosine to Inosine (A-to-I) RNA editing is a post transcriptional, site-specific modification process, catalyzed by Adenosine Deaminase acting on RNA (ADAR) gene family.

ADAR enzymes play an essential role in embryogenesis and normal life. Since ADARs act on double strand RNA, most A-to-I editing occurs within repetitive elements, particularly Alu elements, since these sequences tend to fold and form double strands. Dysregulated ADAR enzymes expression and consequently, altered A-to-I editing patterns were reported in several human diseases, particularly in cancer. Furthermore, reintroduction of correct ADAR expression deteriorates with cancer progression. To further study A-to-I editing role in humans, we compared, in embryonic tissues and respective adult tissues, the editing level of various target genes in coding and non-coding Alu elements. The editing activity in most adult tissue samples was substantially greater than in fetal ones, for most genes tested.

 In human embryonic stem cells (hESC) the level of A-to-I editing decreases during spontaneous differentiation. The knockdown of ADAR1 in hESCs results in elevation of differentiation genes expression suggesting a role for ADAR1 and editing in the regulation of pluripotent stem cells.

To further study this role in early human development, we tried to generate hESC and embryonal carcinoma clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated clones that overexpressed GFP.  Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its transduction into hESCs, the expression levels of this protein decreased dramatically post infection.  These results suggest that ADAR1 protein is substantially regulated in undifferentiated human pluripotent stem cells (hPSC), and that ADAR1-p110 cannot be overexpressed in hPSCs.

We next focused our study on induced pluripotent stem cells (iPSCs). We found that A-to-I editing levels change throughout reprogramming, and that iPS cells editing levels are similar to those of hESCs regardless of the editing level of the reprogrammed source cells. Induction of reprogramming in source cells in which ADAR1 protein level was either down- or upregulated resulted in varied efficiencies of iPSCs colony formation. Furthermore, iPSC clones derived from source cells in which the ADAR1 level was downregulated, lost their iPSC properties shortly after iPSC colonies emerged, and underwent cancerous transformation. Taken together our results suggest a role for A-to-I RNA editing in the regulation of pluripotent stem cells and in the induction and maintenance of early iPSCs.