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Characterization of the function and mechanism of an orphan 3'-5' polymerase implicated in noncoding RNA processing.

Dodbele, Samantha
http://rave.ohiolink.edu/etdc/view?acc_num=osu1574698595845497

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Until the discovery of tRNAHis guanylyltransferase (Thg1) from Saccharomyces cerevisiae (Sc-), nucleotide polymerization was believed to exclusively occur in the 5'-3' direction. Thg1 shifts this paradigm by catalyzing the addition of a required guanosine to the 5'-end of tRNAHis in a 3'-5' direction. Enzymes exhibiting similarity to ScThg1, called Thg1-like proteins (TLPs) catalyze a similar 3'-5' polymerization. TLPs have been found in all three domains of life, including eukaryotic organisms such as Dictyostelium discoideum (Ddi-). However, the roles and mechanisms of TLPs compared to their relatively more well-studied Thg1 counterparts are less understood. Previous work has demonstrated the functions of three Thg1 family enzymes in the model eukaryote D. discoideum. DdiThg1 is responsible for cytosolic tRNAHis maturation, while the TLP enzymes, DdiTLP2, and DdiTLP3 are responsible for mitochondrial tRNAHis maturation, and mitochondrial tRNA 5'-editing, respectively. However, the biological function of a fourth essential Thg1 family enzyme encoded in D. discoideum, DdiTLP4, remains unknown. Now we have the first evidence to suggest that the essential function of DdiTLP4 is due to its role in small RNA processing and its activity on specific ncRNA substrates. Depletion of DdiTLP4 followed by RNA-Seq was used to identify in vivo substrates of DdiTLP4 and enabling the identification of any type of RNA whose 5'-end sequence is altered in the absence of DdiTLP4 activity. Additionally, kinetic investigation reveals that DdiTLP4 terminates 3'-5' nucleotide addition by a distinct mechanism that may be related to its role in small ncRNA processing. The significance in understanding the biological and mechanistic intricacies DdiTLP4 lies in its versatility in how it catalyzes 3'-5' polymerase activity. It is the first Thg1 family enzyme shown to act on small ncRNAs in addition to tRNAs in vitro and thus provides new insights into diverse biological roles and mechanisms of 3'-5' polymerization.
Jane Jackman (Advisor)
Juan Alfonzo (Committee Member)
Ralf Bundschuh (Committee Member)
Karin Musier-Forsyth (Committee Member)
194 p.
Biochemistry
Thg1; ncRNA; Dictyostelium discoideum; RNA-Seq

Recommended Citations

Citations

  • Dodbele, S. (2019). Characterization of the function and mechanism of an orphan 3'-5' polymerase implicated in noncoding RNA processing. [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574698595845497

    APA Style (7th edition)

  • Dodbele, Samantha. Characterization of the function and mechanism of an orphan 3'-5' polymerase implicated in noncoding RNA processing. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1574698595845497.

    MLA Style (8th edition)

  • Dodbele, Samantha. "Characterization of the function and mechanism of an orphan 3'-5' polymerase implicated in noncoding RNA processing." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574698595845497

    Chicago Manual of Style (17th edition)

osu1574698595845497
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This open access ETD is published by The Ohio State University and OhioLINK.