DExD/H-box proteins are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. These proteins are thought to use energy from ATP to facilitate RNA conformational changes and folding transitions, but relatively little is known on a molecular level about how they manipulate RNA structure. While many of the RNA and RNA-protein targets of DExD/H-box proteins are large, complex, and difficult to study in vitro, it was shown in 2002 that the Neurospora crassa CYT-19 protein functions in folding of several mitochondrial group I introns. Further work indicated that CYT-19 can also interact productively with group II introns, indicating that it possesses general RNA chaperone activity, and the related yeast protein Mss116p was shown to function similarly. The goal of this project has been to use the well-defined, tractable system of CYT-19 and group I RNAs to dissect the mechanisms of RNA chaperone activity by DExD/H-box proteins. In addition to increasing knowledge of essential cellular processes, this work has implications for diseases, as DExD/H-box proteins are required for replication of viruses including HCV, and overexpression of human DExD/H-box proteins is linked to colon and prostate cancer. Progress during the current funding period led to a general model for RNA chaperone activity, around which this proposal is centered. First, CYT-19 can disrupt RNA structure indiscriminately, unfolding both the native state and a long-lived misfolded conformer of a non-cognate group I intron with efficiencies that depend on the relative stabilities of the RNA species but not on any specific structural features. However, its activity is not fully indiscriminate;it acts preferentially on structured RNAs by forming an additional 'tethering'interaction, and it unwinds a short helix of a group I intron (P1) only when P1 does not 'dock'into tertiary contacts with the rest of the RNA. These results suggest that CYT-19 may disrupt preferentially misfolded RNAs that cannot pack correctly. The goals of the current proposal are to delve further into the physical basis of RNA chaperone activity by DExD/H-box proteins, to probe for specificity using CYT-19 and its physiological substrates, and to test hypotheses on the mechanisms and implications of tethering and inhibition by tertiary contacts.
Specific Aims are: 1) to use an oligonucleotide displacement assay and directed hydroxyl radical footprinting to probe pathways of RNA chaperone activity on group I introns;2) to determine whether CYT-19 acts on its cognate group I introns with specificity that is absent with a non-cognate RNA;3) to probe further the inhibition by tertiary contact formation, including testing the hypothesis that the inhibition is a general feature of chaperone activity by determining whether it is present for different structural elements and within a different group I intron;and 4) to test the hypotheses that the tethering interaction is formed by a highly basic 'tail'of CYT-19 and related DExD/H-box proteins, and that this interaction can remain intact during local unwinding. Results are expected to guide models for DExD/H-box proteins in all aspects of RNA metabolism.

Public Health Relevance

The goal of this project is to understand how RNA chaperone proteins assist RNAs as they fold to specific structures and exchange between structures. These proteins are required for viral replication and are linked to human cancer, so understanding how they function is important for understanding and ultimately treating human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM070456-06
Application #
7737923
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Preusch, Peter C
Project Start
2004-05-01
Project End
2011-08-31
Budget Start
2009-09-30
Budget End
2010-08-31
Support Year
6
Fiscal Year
2009
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78713
Yangyuoru, Philip M; Bradburn, Devin A; Liu, Zhonghua et al. (2018) The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism. J Biol Chem 293:1924-1932
Gilman, Benjamin; Tijerina, Pilar; Russell, Rick (2017) Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs. Biochem Soc Trans 45:1313-1321
Busa, Veronica F; Rector, Maxwell J; Russell, Rick (2017) The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates. Biochemistry 56:3571-3578
Cannon, Brian; Kachroo, Aashiq H; Jarmoskaite, Inga et al. (2015) Hexapeptides that inhibit processing of branched DNA structures induce a dynamic ensemble of Holliday junction conformations. J Biol Chem 290:22734-46
Russell, Rick (2015) Unwinding the mechanisms of a DEAD-box RNA helicase in cancer. J Mol Biol 427:1797-800
Jarmoskaite, Inga; Bhaskaran, Hari; Seifert, Soenke et al. (2014) DEAD-box protein CYT-19 is activated by exposed helices in a group I intron RNA. Proc Natl Acad Sci U S A 111:E2928-36
Pan, Cynthia; Potratz, Jeffrey P; Cannon, Brian et al. (2014) DEAD-box helicase proteins disrupt RNA tertiary structure through helix capture. PLoS Biol 12:e1001981
Mitchell 3rd, David; Russell, Rick (2014) Folding pathways of the Tetrahymena ribozyme. J Mol Biol 426:2300-12
Russell, Rick; Matouschek, Andreas (2014) Chance, destiny, and the inner workings of ClpXP. Cell 158:479-80
Jarmoskaite, Inga; Russell, Rick (2014) RNA helicase proteins as chaperones and remodelers. Annu Rev Biochem 83:697-725

Showing the most recent 10 out of 38 publications