Project Grant F32GM163387

MOLECULAR MECHANISMS OF TRANSURIDYLTRANSFERASE ACTIVITY AND SUBSTRATE SPECIFICITY - SUMMARY CELLS USE 3' TAGGING OF RNA TO CONTROL THEIR FATE. FOR EXAMPLE, URIDINE TAILS CAN MARK RNA FOR FURTHER PROCESSING OR DEGRADATION. PROPER BIOGENESIS OF LET-7, A MICRORNA THAT REGULATES CELLULAR PROLIFERATION, OFTEN REQUIRES 3' MONOURIDYLATION OF ITS PRECURSOR PRE-LET-7. IN CONTRAST, OLIGOURIDYLATION OF PRE-LET-7 LEADS TO ITS DEGRADATION, ALLOWING THE EXPRESSION OF LET-7'S ONCOGENE TARGETS. BOTH MONO- AND OLIGOURIDYLATION OF PRE-LET-7 ARE CARRIED OUT BY THE SAME ENZYMES, THE REDUNDANT TERMINAL TRANSURIDYLTRANSFERASES (TUTASES) TUT4 AND TUT7 (TUT4/7). PRE-LET-7 OLIGOURIDYLATION REQUIRES THE COFACTOR LIN28A, WHICH IS EXPRESSED IN STEM-LIKE CELL STATES AND CERTAIN CANCERS. TUT4/7 OLIGOURIDYLATE ADDITIONAL SUBSTRATE RNAS OF DISTINCT STRUCTURE AND SEQUENCE FROM PRE- LET-7S, SUCH AS MRNAS WITH SHORT POLYADENYLATED (POLY(A)) TAILS. CURIOUSLY, THIS OCCURS EVEN IN THE ABSENCE OF LIN28. DESPITE TUT4/7'S ESTABLISHED IMPORTANCE FOR CELL IDENTITY, THE EXTENT OF THEIR POTENTIAL SUBSTRATES, AND HOW THEY SELECT THESE SUBSTRATES, REMAINS A KEY QUESTION. THE GOAL OF THIS PROPOSAL IS TO DECIPHER THE MECHANISM OF TUT4/7 ACTIVITY ON DIFFERENT SUBSTRATES THROUGH THE FOLLOWING AIMS: (1) EXAMINE THE MECHANISM OF TUT4/7 URIDYLATION OF POLY(A) SUBSTRATES, AND (2) IDENTIFY ADDITIONAL TUT4/7 SUBSTRATES. CRYSTALLOGRAPHY DATA FOR THE CATALYTIC DOMAIN SUGGEST SIMILARITIES IN THE BASIC URIDYLATION MECHANISM FOR OLIGOURIDYLATED AND POLY(A) SUBSTRATES. THIS HIGHLIGHTS A GAP IN OUR UNDERSTANDING: WHAT ARE THE MOLECULAR DETERMINANTS OF BINDING TO RNAS WITH STRUCTURAL DIFFERENCES AND REQUIREMENTS FOR ACTIVITY (E.G., POLY(A) OLIGOURIDYLATION DOES NOT REQUIRE LIN28A)? AIM 1 WILL FOCUS ON STABILIZING NEAR-FULL-LENGTH TUT4/7 IN COMPLEX WITH POLY(A) SUBSTRATE FOR CRYO-EM STUDIES. PRELIMINARY DATA INDICATE THAT LIMITING A DISORDERED REGION THAT COULD CONTRIBUTE TO PROTEIN INSTABILITY RESULTS IN AN ACTIVE ENZYME THAT CAN BE USED FOR STRUCTURAL STUDIES. ALTERNATIVE APPROACHES INCLUDE MODIFICATIONS TO SAMPLE CONDITIONS AND A SYNTHETIC SUBSTRATE WITH A CHEMICAL TRAP. AIM 2 WILL INVESTIGATE THE EXTENT OF TUT4/7 SUBSTRATES. I WILL USE A CLICK CHEMISTRY STRATEGY TO LABEL, CAPTURE, AND SEQUENCE OLIGOURIDYLATED CELLULAR RNAS. COMBINED, THESE STUDIES WILL REVEAL IMPORTANT CELLULAR RNA REGULATION MECHANISMS RELEVANT TO THE CONTROL OF CELLULAR DIFFERENTIATION AND CANCER PROGRESSION. FOR SUCCESS IN THIS PROJECT, I WILL DRAW ON MY EXISTING EXPERTISE IN PROTEIN BIOCHEMISTRY AND CHEMICAL BIOLOGY, AND BUILD MY STRUCTURAL BIOLOGY SKILLSET. I WILL TRAIN UNDER DR. LEEMOR JOSHUA-TOR, AN EXPERT IN STRUCTURAL CHARACTERIZATION OF NUCLEIC ACID/PROTEIN COMPLEXES, WITH THE SUPPORT OF FIRST-RATE CORE FACILITIES AND EDUCATIONAL RESOURCES AT COLD SPRING HARBOR LABORATORY. THIS WORK WILL PROVIDE A STRONG FOUNDATION FOR MY CAREER IN RESEARCH.