An Initial Characterization of Terminal Uridylyl Transferase 2, a Non-Canonical Poly(A) Polymerase

Abstract

Located at the 3’ end of mRNA transcripts, the poly-adenosine (poly(A)) tail determines if a mRNA will be translated. With a shortened poly(A) tail, mRNAs in the cytoplasm are dormant and prone to degradation. However, mRNAs can be reactivated by non-canonical poly(A) polymerases, which adenylate mRNAs to lengthen their poly(A) tails. The enzyme Tut2 polyadenylates mRNAs yet monoadenylates their antagonist miRNAs, to stabilize both against degradation. Since Tut2’s activation and regulation are not fully known, we endeavored to characterize Tut2 further through solving its apo-crystal structure, to visualize Tut2 by itself. While the structure was not obtained in this study, in developing a soluble construct, we have shown human Tut2 does not require an accessory protein to bind it for activation, as GLD3 was initially thought to do. This implies human Tut2, unlike C. elegans GLD2, is catalytically active on its own. This conclusion was demonstrated in Tut2’s high affinity for RNA and how having functional active site rendered Tut2 insoluble for purification. With the soluble construct developed in this study, future efforts will be directed towards completing the characterization of Tut2 by solving Tut2’s apo-crystal structure.