טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentFremder Ella
SubjectAddition of Poly(A)and Poly(A)-Rich Tails during RNA
Degradation in the Cytoplasm of Human Cells
DepartmentDepartment of Biology
Supervisor Professor Gadi Schuster
Full Thesis textFull thesis text - English Version


Abstract

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Polyadenylation of RNA is a posttranscriptional modification that can play two somewhat opposite roles: stable polyadenylation of the RNA in eukaryote cells contributes to nuclear export, translation initiation, and may also effect transcript longevity. Conversely, transient polyadenylation destines the RNA molecules to rapid exonucleolytic degradation. The latter role has been shown to take place in prokaryotes and organelles, as well as in the nucleus of eukaryotic cells.

In recent years, the barrier dividing these two systems in the biological world had narrowed. Research data from human mitochondria, and also from nuclei of yeast and human cells showed that these two seemingly opposing forms of polyadenylation can co-exist simultaneously within the same cellular compartment. Following this realization, we asked whether transient poly(A) tails are present in the cytoplasm of human cells along with the stable polyadenyaltions. Indeed, our research data presents evidence of hetero- and homopolymeric adenylation of truncated rRNA and mRNA molecules, of both endogenous and viral genes, in the cytoplasm of human cells. Although nucleus/cytoplasm fractionation serves as a valuable means to determine cellular localization, we could not ignore the possibility that truncated RNA molecules could have been adenylated in the nucleus and "leaked" out into the cytoplasm.  Therefore, experiments were designed in order to definitively assess the cytoplasmic localization of truncated, polyadenylated transcripts while identifying their tail nature: homopolymeric or heteropolymeric. We analyzed viral transcripts encoded in the genomes of the Vaccinia and Sindbis viruses. These viruses are exclusively located in the cytoplasm and their components do not enter the nucleus during their entire life cycle; hence, the detection of truncated, polyadenylated RNA transcribed from these viral genomes confirmed cytoplasmic localization.

Moreover, using RNAi to knock down the major RNA degradation machinery, we demonstrated that these adenylated rRNA molecules are indeed degradation intermediates, implicating that a mechanism of RNA decay, involving transient polyadenylation, is present in the cytoplasm of human cells.

Our findings do not only demonstrate that poly(A)-assisted RNA decay exist throughout almost all the organisms and cell compartments, but also, as in the nucleus and mitochondrion, show that stable and transient poly(A) can coexist within a single cellular compartment.