Structural approaches to targeting RNA terminal uridylytransferases

Let-7 microRNA (miRNA) plays a crucial role in the control of cell differentiation and proliferation, which also makes it a suppressor for the occurrence and development of tumors. Pre-let-7 is one of the intermediates in the maturation process of let-7 and two uridylyltransferases - ZCCHC6/TUT7, and ZCCHC11/TUT4 - play essential roles in the downstream processing of let-7 pre-miRNA. In the absence of RNA binding protein Lin28A, they perform 3’ mono-uridylation, leading to pre-let-7 maturation. In the presence of Lin28A, they together form a TUT4(7)-Lin28A-pre-let-7 holo-complex and perform oligo-uridylation, which subsequently leads to exonucleolytic pre-let-7 degradation. However, with no full structural information about TUT4/7, especially the lack of a complex structure with pre-let-7, their detailed uridylation mechanism is still a mystery.
X-ray crystallography and cryo-EM are the mainstream for structure determination and have been developed tremendously in the past decades. This project combined both techniques for structural study of TUT4/7 in apo or complex forms with pre-let-7 in the presence or absence of Lin28A, in order to unravel the details of the uridylation mechanism at atomic level.
With regards to protein purification, I have established and optimized high throughput (HTP) cloning and expression methods for TUT4/7 based on the E.coli system. An optimized two-step purification protocol allows for the purification of both TUT4 and TUT7 in full length or truncated forms. Assays confirm both full length TUT4 and TUT7 are catalytically active. These highly purified proteins are essential milestones on the way to our goal of structural characterization of TUT4 (7) by X-ray crystallography and cryo-EM.
In the case of TUT7, I have determined the crystal structure of the TUT7 catalytic module (CM) at 2.6 Å, which can serve as a basis for fragment screening trials aiming for identifying small molecules with oncogene inhibition potential. With cryo-EM, I reconstructed four high-resolution structures. The dynamic TUT7 full length apo structure is resolved at 4 Å, showing an overall “C-shape” model with two nucleotidyltransferase domains (NTDs) connected by flexible linkers. The 3.7Å binary complex of TUT7 with pre-let-7 reveals the mono-uridylation state, in which the pre-let-7 stem locks onto the CM, stabilizing the dynamic active-site cleft, while the pre-let-7 PRE loop region is close to the let-7 interacting module (LIM) but without a direct interaction or the interaction is transitory/dynamic. The zinc finger regions of the LIM are missing in this complex. Finally, two ternary complex structures were obtained from the TUT7/pre-let-7/Lin28A complex, determined at 3.8 Å and 3.6 Å respectively. The zinc finger in the LIM is visualized in both structures, while in conformation-I, the CM is missing, presumably due to the flexible linker regions connecting the LIM and CM, representing a pre-oligo-uridylation state. The other ternary complex in conformation-II reveals the additional densities of CM and a novel structure of zinc knuckle-1, of which the latter binds to the RNA stem, showing a second activated pre-oligo-uridylation state. The four high-resolution structures presented allow a full description of conformational changes from mono- to oligo-uridylation.
In the study of TUT4, the TUT4 CM crystalised and diffracted to 4.5 Å, indicating the possibility of obtaining well-diffracting crystals for subsequent fragment screening trials (Li, 2020); while the TUT4 LIM in complex with inhibitory nanobody S2A4 diffracted to 5 Å and is under optimization currently. Using cryo-EM, a 3.7 Å ternary complex of a truncated TUT4 (TUT4_miniF)/pre-let-7/Lin28A was determined, showing a similar assembly to one of the models of TUT7/pre-let-7/Lin28A (conformation-I). Furthermore, the cold shock domain of Lin28A was identified in another TUT4_miniF/pre-let-7/Lin28A model solved at 12 Å. The TUT4 full length protein was purified successfully recently and preliminary cryo-EM trials together with pre-let-7/Lin28A show a different shape compared to TUT4_miniF/pre-let-7/Lin28A and close to structure of the TUT7 ternary complex in conformation-II.
The structural work undertaken in this project establishes a strong basis for further understanding the uridylation mechanism at atomic level. This detailed information will provide important details for the rational design and improvement of TUT inhibitors with the potential to improve prospects for cancer patients suffering from a range of malignancies, including some of unmet need.

Keywords: structural studies reveal basis of tumor-suppressor let-7 pre-miRNA  uridylation by TUT enzymes regulated by oncogenic Lin28A
Advisor: Gilbert, R