Structural Studies Of Post-Transcriptional Gene Regulation

National Institute Of Environmental Health Sciences

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Abstract

Gene regulation at the RNA level, or RNA regulation, is a means to rapidly control gene expression for cells in our bodies to properly respond to normal and disease-related stimuli, such as during embryonic development or growth, toxic exposures, and inflammation. This project collects and analyzes data for understanding these processes at the molecular level. The goal of our project is to understand how RNA-binding proteins choose their specific targets for proper gene regulation. Our current work focuses on determining how RNA regulatory proteins act together to control expression of their targets. We used biochemical assays and high-resolution structural methods to understand the functions of proteins that regulate embryonic development and reproduction. We use our molecular understanding to guide biological studies in human cells and model organisms. We also studied the atomic structure and kinetics of enzymes that participate in maturation of transfer RNA (tRNA) and amplification of small interfering RNAs. We are leaders in studies of Pumilio/FBF (PUF) RNA regulatory proteins, which typically repress protein expression when bound to their target RNAs. The human Pumilio proteins are particularly important for fertility, growth, and neurological function, although they are likely to be important for many other processes and diseases. We determined the first crystal structure of a PUF protein bound to RNA, which showed how PUF proteins recognize RNA sequence elements. In the following years we have discovered important details of how a variety of PUF proteins carefully choose the correct RNAs to regulate. In this fiscal year, we published a study of how PUF protein RNA recognition and function require additional ‘partner’ proteins. We found that a partner protein, LST-1, links two FBF-2 PUF proteins on an RNA and this complex of proteins on the RNA is required to fully repress protein expression in germline stem cells and to activate protein expression as cells begin to differentiate. We also studied the ability of additional PUF proteins FBF-1 and PUF-3 to regulate the same genes. This illustrates the importance of redundancy of RNA regulatory proteins and multiple RNA elements in the same gene to preserve fertility: Different proteins can substitute for one another or bind to different RNA elements to assure the correct genes are expressed in the right cells at the right times. This work was done in the model organism C. elegans and has now led to our discovery of similar protein partnerships in human reproduction. These studies are ongoing and an important focus of our future work. In this fiscal year, we have also continued studies of transfer RNA processing enzymes and have obtained biochemical and structural data that establish the unique sequence specificity of two enzymes that generate the essential 3´ tail that carries the amino acids to be incorporated into proteins. Without the correct 3´ tail, protein production (and therefore life) cannot occur. We also determined the kinetic properties of an enzyme that result in the addition of a unique 3´ RNA modification that leads to amplification of small RNAs. This unusual enzyme activity in a model organism has potential to be harnessed for broad applications in human cells.