Structured RNAs and RNA-protein complexes (RNPs) are involved in many essential biological processes and the specific conformations of these RNAs are crucial to their various functions. However, in vitro studies have found that RNA has propensity for misfolding into inactive species that often consist of extensive secondary and tertiary interactions, which can be locally and globally stabilizing, resulting in long-lived non-native conformers. DEAD-box helicases are one class of proteins that have been found to accelerate folding and rearrangements of highly structured RNAs. While these proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt the tertiary interactions that often stabilize both native and misfolded RNA conformations. We used single molecule fluorescence to probe the mechanism by which DEAD-box proteins facilitate global unfolding of a structured RNA. DEAD-box protein CYT-19, a mitochondrial protein from Neurospora crassa, was found to destabilize a specific tertiary interaction with the Tetrahymena group I intron ribozyme using a helix capture mechanism. The protein molecule binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then uses ATP to unwind the helix, liberating the product strands. Ded1, a multi-functional DEAD-box protein found in Saccharomyces cerevisiae, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively-folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome. / text

The DEAD-Box RNA helicase family is a conserved group of enzymes that function in gene expression through ATP-dependent RNA unwinding and ribonucleoprotein (RNP) remodeling. DEAD-Box helicases function in multiple cellular processes, including pre-mRNA processing, translation, mRNA export, and mRNA decay. Although DEAD-Box proteins are critical for gene expression, much of their mechanistic activities are poorly understood. DEAD-Box proteins have increasingly been linked to tumorigenesis in humans, and better defining their activity at the mechanistic level will aid in understanding the underlying disease pathology. In this study, we used the model organism Saccharomyces cerevisiae to study the human DEAD-Box protein DDX3 orthologue, Ded1, and its role in translation initiation during cellular stress. Recently, we have found that Ded1 is an important mediator of the cellular stress response in a TOR-dependent manner. TOR regulates protein synthesis dependent on energy availability in the cell by regulating the assembly of the eukaryotic translation initiation complex. Human DDX3 has been found to interact with translation initiation complex subunit eIF4E and Ded1 has been found to interact with the translation initiation complex subunit eIF4G. In this study, we examined the purported interaction region between Ded1 and eIF4G on the C-terminus of Ded1 and found that ded1 Δ591-604 prevents eIF4G degradation under rapamycin treatment and confers resistance to rapamycin-induced growth inhibition. We also examined putative regulatory phosphorylation sites in the purported Ded1 eIF4G binding region. We propose that the Ded1/eIF4G interaction is critical for the repression of translation by Ded1 and that eIF4G degradation may be regulated by Ded1 under stress conditions.

RNA helicases are found in all kingdoms of life, functioning in all aspects of RNA biology mainly through modulating structures of RNA and ribonucleoprotein (RNP) complex. RNA structures have fundamental impacts on steps in gene expression, including transcription, pre-mRNA processing, and translation. However, the precise roles and regulatory mechanisms of RNA structures in co- and post-transcriptional processes remain elusive. By probing genome-wide RNA structures in vivo, a recent study suggested that ATP-dependent factors, such as RNA helicases, maintain the actively unfolded state of RNAs. Among all RNA helicases, DEAD-box proteins form the largest family in eukaryotes, and have been shown to remodel RNA/RNP structures both in vitro and in vivo. Nevertheless, for the majority of these enzymes, it is largely unclear what RNAs are targeted and where they modulate RNA/RNP structures to regulate co-transcriptional processes. To fill the gap, my research focused on identification of the RNAs and structures targeted by the DEAD-box protein Dbp2 in S. cerevisiae to uncover the cellular processes that Dbp2 is involved in.<br><div><div>My studies revealed a role of Dbp2 in transcriptional termination. Dbp2 binds to ~34% of yeast mRNAs and all snoRNAs, and loss of DBP2 leads to a termination defect as evidenced by RNA polymerase II (RNAPII) accumulation at 3’ ends of these genes. In addition, the binding pattern of Dbp2 in mRNAs is highly similar to Nrd1 and Nab3 in the Nrd1-Nab3-Sen1 (NNS) termination complex, and deletion of DBP2 leads to reduced recruitment of Nrd1 to its target genomic loci. In Dbp2 and NNS targeted 3’ UTRs, RNA structural changes resulted from DBP2 deletion also overlap polyadenylation elements and correlate with inefficient termination, and loss of stable structure in the 3’ UTR bypasses the requirement for Dbp2. These findings lead to a model that Dbp2 promotes efficient termination of transcription through RNA structure remodeling.</div><div>Interestingly, my research also revealed the requirement of DBP2 for efficient splicing, as loss of DBP2 leads to accumulation of unspliced pre-mRNAs. Moreover, this function is dependent on the helicase activity of Dbp2. Further studies are needed to characterize the molecular mechanism of how Dbp2 facilitates splicing in cells. Overall, my research demonstrated that DEAD-box RNA helicases remodel mRNA structure in vivo and that structural alteration can be essential for proper gene expression.</div></div>

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RNAs associate with chromatin through various ways and carry out diverse functions. One mechanism by which RNAs interact with chromatin is by the complementarity of RNA with DNA, forming a three-stranded nucleic acid structure named R-loop. R-loops have been shown to regulate transcription initiation, RNA modification, and immunoglobulin class switching. However, R-loops accumulated in the genome can be a major source of genome instability, meaning that they must be tightly regulated. This thesis aims to identify R-loop-binding proteins systemically and study their regulation of R-loops. Using immunoprecipitation of R-loops followed by mass spectrometry, with or without crosslinking, a total of 364 proteins were identified. Among them RNA-interacting proteins were prevalent, including some already known R-loop regulators. I found that a large fraction of the R-loop interactome consists of proteins localized to the nucleolus. By examining several DEAD-box helicases, I showed that they regulate rRNA processing and a shared set of mRNAs. Investigation of an R-loop-interacting protein named CEBPZ revealed its nucleolar localization, its depletion caused down-regulation of R-loops associated with rRNA and mRNA. Characterization of the genomic distribution of CEBPZ revealed its colocalization with insulator-regulator CTCF. When studying if CEBPZ recruits CTCF, I found that instead of regulating CTCF binding, CEBPZ depletion has a major effect on the performance of CUT&RUN, a technique for identifying DNA binding sites of proteins. How CEBPZ affects CUT&RUN is still under investigation, the study of which may help us understand the roles of CEBPZ in regulation of global chromatin structure and genome integrity.

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