DEAD-box proteins are superfamily 2 RNA helicases that are required for virtually every process carried out by structured RNAs, from pre-mRNA splicing and translation to intracellular trafficking of proteins and RNAs. They are also required for replication of viruses including HIV-1 and HCV and overexpression is linked to colon and prostate cancer. These proteins use energy from ATP binding and hydrolysis to facilitate RNA conformational changes and folding transitions, but we have limited molecular knowledge about how they manipulate RNA structure. Insights from mechanistic studies will be critical for deep understanding of fundamental biological processes and for understanding and ultimately treatment of important viral diseases and cancer. We have focused on the fungal DEAD-box proteins CYT-19 and Mss116p, which function as general RNA chaperones in folding of mitochondrial group I and group II self-splicing introns. These systems are powerful for mechanistic studies because the RNAs are relatively simple and tractable, and their catalytic activity provides a robust and sensitive readout for formation of the native state. In years 1-5 of funding (2004-2009), we used a well-studied group I intron from Tetrahymena thermophila to show that CYT-19 functions as a true chaperone, facilitating refolding of a long-lived misfolded conformation without functioning in the downstream catalytic steps. We found that CYT-19 disrupts structure non-specifically, without distinguishing native from misfolded conformations, and this activity favors accumulation of the native RNA because it is much more stable than the misfolded conformation. Since submitting a renewal that received two years of ARRA funding (2009-2011), we made further advances. Single molecule fluorescence and rapid kinetics showed directly that CYT-19 can be strongly inhibited by tertiary structure, unwinding a short helix only after the helix spontaneously 'undocks'from tertiary contacts with the intron core. Rapid kinetics experiments also supported a model in which a C-terminal 'tail'of CYT-19 contacts structured RNA and tethers the helicase core to disrupt nearby structure, and a series of rate measurements indicated that CYT-19 completely unwinds short RNA helices in a single cycle of ATP- dependent conformational changes. Together, the work leads to a general model for RNA chaperone activity by DEAD-box proteins in which the proteins are localized to structured RNAs by tethering, and they disrupt exposed elements of secondary structure non-processively to allow these segments an opportunity to form new contacts. Here we propose to test and extend this model in two important directions.
In Aims 1 and 2, we will use simple duplex substrates and an arsenal of experimental approaches to probe how the ATPase cycle is coupled to RNA unwinding and to what extent tethering constrains the position and orientation of the helicase core.
In Aims 3 and 4 we will use physical and chemical approaches to follow folding of group I and group II introns that rely on CYT-19 and Mss116p in vivo, testing specific hypotheses and probing whether our model for general chaperone activity describes the effects of these proteins on folding of their cognate RNAs.
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.
|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|
|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|
|Jarmoskaite, Inga; Russell, Rick (2014) RNA helicase proteins as chaperones and remodelers. Annu Rev Biochem 83:697-725|
|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; 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|
|Cannon, Brian; Pan, Cynthia; Chen, Liangjing et al. (2013) A dual-mode single-molecule fluorescence assay for the detection of expanded CGG repeats in Fragile X syndrome. Mol Biotechnol 53:19-28|
|Cannon, Brian; Kuhnlein, Jeffrey; Yang, Soo-Hyun et al. (2013) Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET. Proc Natl Acad Sci U S A 110:18868-73|
|Mitchell 3rd, David; Jarmoskaite, Inga; Seval, Nikhil et al. (2013) The long-range P3 helix of the Tetrahymena ribozyme is disrupted during folding between the native and misfolded conformations. J Mol Biol 425:2670-86|
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