Mechanistic and Functional Studies of RNA Processing Pathway Components

Abstract

The delicate interplay between proteins and the RNAs that they bind or affect forms the bases for numerous, coordinated and interconnected RNA metabolic pathways that maintain all cellular life. The pervasiveness and diversity of protein-RNA interactions, puts RNA binding or processing proteins in integral regulatory positions, the disregulation of which, underlie major human diseases. Here, I have examined three components of major RNA processing pathways, to gather insight on their diverse mechanisms of action. The Intron degradation pathway is responsible for maintaining cellular free nucleotide levels, which function as sources of energy, signaling molecules and precursors for DNA and RNA synthesis. In this dissertation, I present the highest resolution X-ray crystal structure and cofactor specificity analysis of the Dbr1 enzyme, which cleaves 2’-5’ phosphodiester bonds within all excised intronic lariats, thus, initiating intron turnover. MicroRNAs are small noncoding RNAs that regulate gene expression; they are themselves processed via a multi-step and multi-component biogenesis pathway. One of the major functions of the oncoprotein LIN28A is to inhibit the biogenesis of let-7 miRNAs by binding and preventing processing by Dicer, and subsequently initiating degradation. Here, I use a comparative approach to understand newly defined interactions between LIN28 and RNAs throughout the transcriptome. The DEAD-Box Helicase P72, on the other hand, participates as an accessory factor to the Microprocessor complex, and enhances processing of certain classes of miRNA. In this work, I characterize a newly observed behavior of P72 that may define new cellular functions associated with RNA binding.