DEAD-box RNA helicases: in vivo roles, protein partners, RNA substrates and mechanistic details – HeliDEAD

The HeliDEAD project consists of eight tasks: (1) single-molecule magnetic tweezers, (2) yeast genetic analyses, (3) protein partner identification, (4) protein partner interactions, (5) enzymatic characterizations, (6) RNA substrate identification, (7) yeast cellular extract assays, and (8) protein cellular localization. In the first task, Partner 2 (Croquette group) and Partner 1 (Tanner group) are using single-molecule magnetic tweezers to investigate the molecular mechanism of RNA helicases. A DNA containing a large hairpin is attached to a glass slide on one end and a magnetic bead on the other. This hairpin is unwound when a magnet is applied and blocked from reforming by a hybridized RNA oligonucleotide. They can then add an RNA helicase and ATP and follow the rate of hairpin reformation under different conditions. Thus, they can tease out the molecular details of the reaction mechanism.
Tasks 2-7 are highly interconnected. Partner 1 is using pull-down experiments of yeast extracts, mass spectroscopy and Western blots to identify the protein partners of RNA helicases. They use yeast genetic to determine if there is functional relationship between the helicases and the identified proteins. For example, they can test to see if over-expression of the protein complements a growth defect of a mutated helicase. They clone the genes of interest into bacterial expression vectors, express and purify the proteins, and then test to see they interact with the helicase or affect its enzymatic activity.
Task 8 is done in association with Partner 3 (N. Belgareh-Touzé) and Partner 1. They are using fluorescence-tagged proteins to elucidate the cellular location of RNA helicases under different growth conditions and to colocalization of potential protein partners.

We recently publish a comprehensive article on tasks 2–5 and 8 in association with Partner 3 (Senissar et al, 2014, Nucleic Acids Res. 42, 10005–10022). In essence, we find that Ded1 is part of the protein complexes that form on the 5' 7-methyl guanosine caps of mRNAs, and we find that Ded1 actively shuttles between the cytoplasm and nucleus. We used pull-down experiments of the Ded1-associated complexes in yeast extracts and mass spectroscopy to identify protein partners. Other factors were identified by Western blot analyses of the isolated complexes. We cloned the genes encoding for these partners, expressed them in E. coli with affinity tags, purified them and then used them to show direct physical interactions with Ded1. We used GFP-tagged Ded1 and various nuclear export mutants to show that Ded1 actively shuttles between the cytoplasm and the nucleus using both the mRNA Mex67 pathway and the exportin-depend pathway involving Crm1. In résumé, we show that in the cytoplasm, Ded1 forms physical and genetic links with eIF4E, Pab1 and eIF4G. In the nucleus, it forms physical and genetic links with Cbp20, Cbp80 and Nab2. Moreover, we showed that the enzymatic activity of Ded1 is moderately stimulated by the presence of these partners. These experiments are gratifying because they are consistent with what others obtain for the mammalian homolog of Ded1, DDX3, which is associated with viral replication and oncogenesis in human diseases. This is ongoing work, and we have subsequently identified additional partners that we are currently characterizing. We have also obtained a mutant of Ded1 that fixes the ATP and RNA ligands with high affinity but that is unable to hydrolyze the ATP and recycle the complexes. Thus, the Ded1 mutant is locked at an intermediate step. We have used this mutant to show that Ded1 accumulates in cellular RNA granules, called P bodies, that are dependent on the presence of certain protein partners and on the cellular state.

RNA helicases, and DEAD-box proteins in particular, are fundamental to genetic expression in all organisms, from simple bacteria to complex multicellular organisms that include humans. These proteins are ATP-dependent molecular motors that have been fine tuned to have very specific roles in all processes involving RNA, from transcription, splicing, export, translation to degradation. Yeast has at least 45 RNA helicases and 25 DEAD-box proteins. These proteins are often essential and rarely interchangeable. Thus yeast provides a useful model system for understanding how RNA helicases work. The well-developed yeast genetic system means that essential proteins can be deleted and replaced with plasmid-borne copies. We can easily make mutations that we can assay in vivo for their ability to support growth and in vitro with purified proteins to determine their enzymatic properties. We can similarly assay genetic links between potential protein partners and characterize the interactions in vitro with the purified components. Finally, yeast provides a simplified platform for studying RNA helicases in metazoans and plants because it typically contains only a single example of each helicase-dependent process, because the expressed proteins are less extensively modified and consequently easier to express and purify, and lastly because the functional conservation means that proteins from higher organisms can be readily tested against their yeast equivalents.
In the future we hope to take what we learned to modify and control the activity of RNA helicases, especially in how they relate to human diseases. For example, hippuristanol is an allosteric inhibitor of RNA binding specifically to eIF4A, which is a DEAD-box protein that is highly expressed during ocogenesis. We have similarly found compounds that inhibit DEAD-box proteins from the infectious trypanosomatid protozoa Leishmania. We are similarly investigating inhibitors of the opportunistic pathogen Candida albicans.

Senissar, M., Saux, A. L., Belgareh-Touzé, N., Adam, C., Banroques, J., & Tanner, N. K. (2014). The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus. Nucleic Acids Res., 42, 10005–10022. doi:10.1093/nar/gku584
Saurabh, R., Bagchi, D., Fiorini, F., Le Hir, H., Tanner, K., Banroques, J., & Croquette, V. (2014) «RNA Helicases on the Move.« Biophys. J., 106, 71a–72a.
Banroques, J. & Tanner, N. K. (2015) Methods to study the structural and functional elements of DEAD-box RNA helicases. Methods Mol. Biol. 1259, 199-209.
Bizebard, T., & Dreyfus, M. (2015) A FRET-based, continuous assay for the helicase activity of DEAD-box proteins. Methods Mol. Biol. 1259, 165-181.

The DEAD-box family of RNA helicases represents a ubiquitous collection of proteins that are found in all three kingdoms of life and that are associated with all processes involving RNA, from transcription to RNA decay. These proteins are often implicated in very specific cellular processes, they are often essential and they are rarely interchangeable. Moreover, some of these proteins are implicated in various pathologies and infectious diseases. Nevertheless, the purified proteins show little or no substrate specificity and no regulation. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. Many of the proteins can displace weak RNA duplexes in vitro, but typically inefficiently. The proteins also are able to displace bound proteins (RNP remodeling), remodel RNA secondary and tertiary structures (RNA chaperones), and to assemble in ribonucleoprotein complexes (RNP assembly). Despite this, little is known about the actual molecular mechanisms of these proteins nor the actual substrates upon which they act. The specificity and regulation of these proteins in vivo must be conferred by the context in which they appear and hence on their protein partners. Hence, our objectives in this proposal are: i) to identify protein partners; ii) to identify the authentic RNAs substrates; iii) to reconstitute functional nucleoprotein complexes in vitro; iv) elucidate the cellular roles; and v) to use single-molecule magnetic tweezers to investigate the molecular mechanism(s) of the proteins.

These experiments are possible because we use the yeast Ded1 protein as a model system; it is an essential protein in vivo that has one of the highest enzymatic activities in vitro. As a consequence, we are the first to develop an assay to measure DEAD-box proteins unwinding duplexes at a single molecule level. This sensitive assay uses single-molecule magnetic tweezers, developed by Vincent Croquette, to easily measure off rates, binding affinities and the effects of cofactors, without the side effects of the applied force. We purify complexes containing Ded1 and determine their compositions by immunodetection and mass spectroscopy. We use yeast genetics to identify and characterize potential partners. We use biochemical and enzymatic approaches to characterize the properties of the different factors. We have already implicated new partners and new RNPs of Ded1. We will use CRAC and other technologies to isolate and characterize the RNA substrates. We will use cellular extracts to characterize the biological role(s) of the protein. Lastly, we are determining the cellular location of the protein under different growth conditions in association with an experienced cellular biologist, Naïma Belgareh-Touzé. We are the first to demonstrate a nuclear location of Ded1.

This proposal brings together three laboratories with very different expertise to create a synergistic team to better understand the cellular roles and molecular mechanisms of DEAD-box helicases.