טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentYara Eid Mutlak
SubjectThe Ribonucleolytic Activity of Human P53
DepartmentDepartment of Biology
Supervisor Full Professor Schuster Gadi
Full Thesis textFull thesis text - English Version


Abstract

The human P53 protein is most commonly known for its fundamental role of a tumor suppressor protein that accumulates in response to DNA damage, oncogene activation, and other stresses. It acts as a nuclear transcription factor to regulate the expression of gene products involved in multiple cellular pathways. Aside from its’ well characterized transcription-dependent activities, P53 harbors additional functions. One such activity is the degradation of ssDNA in a 3’ to 5’ exonucleolytic manner, revealing a possible direct involvement of the P53 protein in the process of DNA damage response and repair.

The human cell produces a complex orchestra of multiple RNA degrading enzymes (RNases), to ensure the correct accumulation of the RNA molecules, as well as the rapid degradation of the incorrect and aberrant transcripts.  RNA molecules with defects in processing, folding, or assembly with proteins are identified and rapidly degraded by the surveillance machinery to prevent malignancies from developing. There are two types of ribonucleases; exoribonucleases which remove nucleotides starting at either the 5’ or 3’ end, and endoribonucleases, which cleave the RNA internally.

Our hypothesis is that human tumor suppressor P53 holds, amongst many other functions, a ribonucleolytic activity.  The primary aim of this research is to explore and characterize the mode of activity of this new P53 ribonuclease. To do so, we first expressed and purified the recombinant protein in E. coli and then performed ribonucleolytic activity assays.

We show that the recombinant human P53 (rP53) displays a ribonucleolytic activity and is capable of degrading ssRNA substrates. rP53 acts as a robust endoribonuclease, yet harbors in addition a residual 3’ to 5’ exoribonuclease activity. Moreover, we unravel a potential, non-exclusive preference of the P53 ribonuclease activity to certain RNA sequences and its’ sensitivity to secondary structured and dsRNA substrates. Histidine number 1 1 5 was found to be important for both DNA and RNA degradation activities, suggesting the activity sites for both these activities are in closed vicinity or perhaps identical. These findings emphasize the importance of further studying and characterizing the new ribonucleolytic activity of the P53, and how it relates to the previously known functions of the protein, unravelling the function of the RNA degradation activity with that of tumor suppressor.