KNOCK OUT AND FUNCTIONAL ANALYSIS OF ENZYMES RESPONSIBLE FOR 3’ OLIGOURIDYLATION AND METABOLISM OF HISTONE MRNA.

Metazoans regulate histone protein synthesis with DNA replication in S-phase via post-transcriptional modification of the histone mRNAs, which see a 30-fold increase in expression at the beginning of S-phase. Histone mRNAs are the only mRNAs that do not end in polyadenylation, but instead in a conserved stemloop. This stemloop, along with Stemloop Binding Protein (SLBP), are required for the unique pathway for histone mRNA processing, translation, and degradation. The stemloop also serves to bind 3’ histone exonuclease (3’hExo), which removes 2nt from the 3’ termini, leaving the stemloop and a 3nt tail. Previous work has shown that if 3’hExo degrades too far past those 2nt, a uridyl transferase (TUT7) will add nontemplated uridines to compensate length back to 3nt past the stemloop. This oligouridylation serves to maintain a proper length for stable histone mRNAs along with longer uridine tails (3+nt) being found during degradation of the histone mRNA. In this thesis, I have refined a deep sequencing strategy to study the 3’ termini of histone mRNA molecules with a single nucleotide resolution of non-templated nt additions. I have also generated CRISPR mediated Knock Out (KO) human cancer cell lines for both TUT7 and 3’hExo. I have analyzed these KO cells with the above deep sequencing strategy, along with flow cytometry and molecular biology, to demonstrate that both 3’hExo and TUT7 are essential to regulating the 3’ termini of stable histone mRNA molecules. I also demonstrate that 3’hExo is involved in initiation of histone mRNA degradation, and that TUT7 helps initiate and progress 3’ to 5’ degradation of histone mRNA. I also demonstrate with flow cytometry and northern blots that 3’hExo KO cells have delayed mitosis, along with inhibited histone mRNA degradation; and TUT7 KO cells have normal cell cycle progression with slightly delayed histone mRNA degradation after S-phase ends. Finally, I show that stimulated degradation of histone mRNA with hydroxyurea is closely mimicked by the natural end of S-phase degradation of histone mRNA.Doctor of Philosoph