PSEUDOURIDINE MODIFICATIONS IN HUMAN tRNAs AND ARCHAEAL rRNAs

Author

Manisha Deogharia, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2018

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Gupta, Ramesh

Abstract

AN ABSTRACT OF THE DISSERTATION OF MANISHA DEOGHARIA, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry presented on May 16, 2018, at Southern Illinois University, Carbondale TITLE: PSEUDOURIDINE MODIFICATIONS IN HUMAN tRNAs AND ARCHAEAL rRNAs MAJOR PROFESSOR: DR. RAMESH GUPTA RNAs undergo several post-transcriptional modifications inside the cell. The most abundant modification found in RNA is pseudouridine. Pseudouridine is present in all major classes of RNA. The classical TΨC sequence of tRNA reflects T (ribothymidine or 5-methyluridine) at position 54 in most Bacteria and Eukarya, and Ψ and C at positions 55 and 56, respectively, in nearly all tRNAs. TrmA and TruB homologs produce T54 and Ψ55, respectively, in Bacteria and Eukarya. However, archaeal tRNAs commonly have Ψ54 (or m1Ψ54) instead of T54, and Pus10 produces both Ψ54 and Ψ55 in these tRNAs. The pus10 gene is present in nearly all Archaea and most eukaryotes, but not in Bacteria and yeast. This coincides with the presence of Ψ54 in archaeal tRNAs and certain tRNAs (for Gln, Trp, Pro Thr, etc.) of animals, and its absence in the tRNAs of Bacteria and yeast. tRNAs for Trp and Pro that function as primers for replication of retroviruses also contain Ψ54. We found that Pus10 is the Ψ54 synthase in eukaryotes. The Ψ54 activity is specific for certain tRNAs, and it requires a conserved Am1AAU sequence at positions 57-60 of the tRNA for its maximum activity. Recombinant Pus10 can also form Ψ54 in select tRNAs and presence of m1A at position 58 is necessary for its maximum activity. Humans have two paralogs of TruB, TruB1, and TruB2 which are predicted to be the Ψ55 synthases for cytoplasmic and mitochondrial tRNAs, respectively. We found that recombinant human Pus10 can also modify Ψ55 of tRNAs in vitro. This Ψ55 activity of human Pus10 is not selective for specific tRNAs. Another pseudouridine synthase, Cbf5, which functions in guide dependent manner, is necessary for Ψ production in 23S rRNA of H. volcanii. Cbf5 is the catalytic component of the box H/ACA ribonucleoprotein complex that brings about these modifications. It consists of a guide RNA and three core proteins Nop10, Gar1, and L7Ae along with Cbf5. We found that Nop10 is necessary for Ψ production in 23S rRNA.