In Vitro Reconstitution of Argonaute poly(ADP-ribosyl)ation and Its Impact on microRNA Activity
Weltzer, Ryan R
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MicroRNA-mediated gene regulation is an important component of cell biology, involved in nearly all cellular processes. MicroRNAs are ~22 nucleotide non-coding RNAs that recognize complimentary mRNA targets and prevent them from being translated. To achieve this, the microRNA (miRNA) must be accompanied by one or more proteins known collectively as the RNA-induced silencing complex (RISC). The central component of the RISC is one of four Argonaute proteins. One of these proteins, Ago2, has endonuclease activity. Guided by a miRNA, Ago2 can directly cleave the mRNA target without assistance from any other RISC members. MiRNA repression is reduced upon stress when all Argonaute members, including Ago2, are modified by poly(ADP-ribose). Similar reduction in microRNA repression occurs upon knockdown of poly(ADP-ribose) degrading enzyme PARG or, conversely, by overexpression of specific poly(ADP-ribose) polymerases including PARP-12 and PARP-13. Moreover, Ago2 has been shown to be associated with PARP-5a, PARP-12 and PARP-13 by co-immunoprecipitation and co-localization at stress granules. The aim of this project is to use recombinant proteins to ADP-ribosylate Ago2 in vitro to determine whether and how this modification affects Ago2-mediated RNA activity and if the modification can be reversed by PARG or another poly(ADP-ribose) hydrolase, ARH3. This thesis will describe the purification of ARH3 and the catalyctic domain of PARP-12 from E. coli. ARH3 is demonstrated to be an active glycohdrolase and PARP-12cat is demonstrated to be a mono(ADP-ribose) tranferase. Our results suggest that Ago2 can be mono(ADP-ribosyl)ated by PARP-12cat and may be poly(ADP-ribosyl)ated by PARP-5a in vitro. Finally, an Ago2 cleavage assay has been recapitulated and optimized. The work in this thesis project has established the necessary tools to test the hypothesis that mono(ADP-ribosyl)ation or poly(ADP-ribosyl)ation will repress Ago2 activity and whether its activity can be recovered with PARG or ARH3. Other future experiments may elucidate the role of PARP-13.