RNA molecules play myriad essential roles in gene expression, yet for many cellular and viral processes, little is known about how RNA complexes coordinate and regulate these dynamic events. This project will apply biophysical approaches aimed at revealing how RNA interactions drive important biological processes such as pre-mRNA splicing and translational frameshifting. Pre-mRNA splicing is catalyzed by the spliceosome, a large and highly dynamic assembly of 5 RNAs and over 100 proteins. We will investigate how spliceosomal RNAs and their associated complexes interact during spliceosome assembly, a process involving large-scale RNA structural rearrangements that ultimately lead to the formation of the spliceosome, a massive ribonucleoprotein particle twice the size of a ribosome. There are no high-resolution structures of the spliceosome, and very little is known at the molecular level about the steps leading to its assembly and activation. Translational frameshifting is another RNA-mediated process that we aim to study and for which there is no high-resolution structural information available. Frameshifting is the process by which ribosomes are directed into an alternate reading frame to synthesize a different protein. Most retroviruses utilize translational frameshifting in the form of an RNA programmed -1 frameshift, which increases the viral genomic coding capacity and serves to regulate the expression of essential genes. This proposal will examine HIV-1 frameshifting and will measure for the first time the relationship between HIV-1 mRNA thermodynamic stability and frameshift efficiency in vivo. We will also explore how frameshifting is linked to RNA packaging in HIV and will test novel, high affinity compounds that bind to the HIV-1 frameshift site, stimulate frameshifting and inhibit HIV replication. Using the tools we have developed for HIV, we will expand these investigations to study the structural basis for +1 reading frame selection, using the well-studied Israeli Acute Paralysis Virus internal ribosome entry site as a model system. These studies will significantly advance our understanding of how mRNAs program translational recoding in human cells, and may eventually lead to the development of novel antiviral therapies. Finally, we will capitalize on a recent breakthrough to explore an exciting new direction aimed at understanding how angiogenin stimulates formation of blood vessels. Angiogenin is a ribonuclease that has been recently found to specifically bind to a non-coding RNA in the nucleolus in order to activate transcription of rRNA, the first step in inducing cellula proliferation.
The research described in this proposal will reveal the structure and function of non-coding RNAs that are essential for normal gene expression and human health. The project will also reveal how viruses such as HIV-1 utilize RNA structures for regulating gene expression and will explore whether these RNA structures can be targeted with small molecules that inhibit viral replication.
| Montemayor, Eric J; Didychuk, Allison L; Yake, Allyson D et al. (2018) Architecture of the U6 snRNP reveals specific recognition of 3'-end processed U6 snRNA. Nat Commun 9:1749 |
| Didychuk, Allison L; Butcher, Samuel E; Brow, David A (2018) The life of U6 small nuclear RNA, from cradle to grave. RNA 24:437-460 |
| Nomura, Yuichiro; Roston, Daniel; Montemayor, Eric J et al. (2018) Structural and mechanistic basis for preferential deadenylation of U6 snRNA by Usb1. Nucleic Acids Res 46:11488-11501 |
| Slosarek, Erin L; Schuh, Amber L; Pustova, Iryna et al. (2018) Pathogenic TFG Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking and Axon Fasciculation. Cell Rep 24:2248-2260 |
| Didychuk, Allison L; Montemayor, Eric J; Carrocci, Tucker J et al. (2017) Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities. Nat Commun 8:497 |
| Montemayor, Eric J; Didychuk, Allison L; Liao, Honghong et al. (2017) Structure and conformational plasticity of the U6 small nuclear ribonucleoprotein core. Acta Crystallogr D Struct Biol 73:1-8 |
| Butcher, Samuel E; Jan, Eric (2016) tRNA-mimicry in IRES-mediated translation and recoding. RNA Biol 13:1068-1074 |
| Rodgers, Margaret L; Didychuk, Allison L; Butcher, Samuel E et al. (2016) A multi-step model for facilitated unwinding of the yeast U4/U6 RNA duplex. Nucleic Acids Res 44:10912-10928 |
| Garcia-Miranda, Pablo; Becker, Jordan T; Benner, Bayleigh E et al. (2016) Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity. J Virol 90:6906-6917 |
| Didychuk, Allison L; Montemayor, Eric J; Brow, David A et al. (2016) Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly. Nucleic Acids Res 44:1398-410 |
