RNA Duplex Unwinding Research Articles

Deep transcriptome profiling reveals limited conservation of A-to-I RNA editing in Xenopus

BackgroundXenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored.ResultsHere, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals.ConclusionsCollectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal’s unique physiology or environmental adaptation.

Structural basis for RNA-duplex unwinding by the DEAD-box helicase DbpA.

DEAD-box RNA helicases are implicated in most aspects of RNA biology, where these enzymes unwind short RNA duplexes in an ATP-dependent manner. During the central step of the unwinding cycle, the two domains of the helicase core form a distinct closed conformation that destabilizes the RNA duplex, which ultimately leads to duplex melting. Despite the importance of this step for the unwinding process no high resolution structures of this state are available. Here, I employed nuclear magnetic resonance spectroscopy and X-ray crystallography to determine structures of the DEAD-box helicase DbpA in the closed conformation, complexed with substrate duplexes and single-stranded unwinding product. These structures reveal that DbpA initiates duplex unwinding by interacting with up to three base-paired nucleotides and a 5' single-stranded RNA duplex overhang. These high-resolution snapshots, together with biochemical assays, rationalize the destabilization of the RNA duplex and are integrated into a conclusive model of the unwinding process.

Step-by-step binding and unwinding of RNA by a Ded1p trimer revealed by combined high-resolution tweezers and fluorescence measurements.

The DEAD-box family of RNA helicases is the largest of all known RNA helicase families, where they remodel RNA by binding and unwinding duplex RNA via an ATP-dependent mechanism. Lacking translocation capability, these helicases are known to unwind only a limited amount, <10–20 bp, locally nearby the site of binding. However, some DEAD-box RNA helicases have recently been shown to unwind efficiently upon oligomerization. The DEAD-box RNA helicase Ded1p has been observed to efficiently unwind RNA duplexes possessing a single-stranded RNA (ssRNA) tail as a trimer. The detailed mechanism of Ded1p trimer assembly and unwinding of RNA remains unknown. We have used combined high-resolution tweezers and single-molecule fluorescence spectroscopy in order to directly observe the detailed step-by-step binding and dissociation of Ded1p protomers and subsequent unwinding and rezipping of duplex RNA. The overall trimer activity previously observed in ensemble experiments was recapitulated. A complete general model of unwinding is developed whereby unwinding is initiated by binding of a single Ded1p to the duplex adjacent ssRNA tail. Subsequently, in sequence two additional Ded1p bind and unwind a 5-7 bp portion of the duplex, each resulting in the full 16 bp duplex unwinding. The reaction is highly dynamic and stochastic, with unwinding combining with frequent rezipping reversals. Rezipping reversals are suppressed when ATP hydrolysis is suppressed via use of an ATP analog. While unwinding and rezipping are easily captured by high-resolution tweezers, Ded1p binding and dissociation on ssRNA tail was measured via the protein-induced-fluorescence-enhancement (PIFE) signal from the fluorophore-labeled tail. The combined methods allowed us to fully observe the coordinated binding and unwinding of the RNA substrate by individual protomers of the Ded1p trimer.

Determination of rate constants for conformational changes of RNA helicases by single-molecule FRET TIRF microscopy.

RNA helicases couple nucleotide-driven conformational changes to the unwinding of RNA duplexes. Interaction partners can regulate helicase activity by altering the rate constants of these conformational changes. Single-molecule FRET experiments on donor/acceptor-labeled, immobilized molecules are ideally suited to monitor conformational changes in real time and to extract rate constants for these processes. This article provides guidance on how to design, perform, and analyze single-molecule FRET experiments by TIRF microscopy. It covers the theoretical background of FRET and single-molecule TIRF microscopy, the considerations to prepare proteins of interest for donor/acceptor labeling and surface immobilization, and the principles and procedures of data analysis, including image analysis and the determination of FRET time traces, the extraction of rate constants from FRET time traces, and the general conclusions that can be drawn from these data. A case study, using the DEAD-box protein eIF4A as an example, highlights how single-molecule FRET studies have been instrumental in understanding the role of conformational changes for duplex unwinding and for the regulation of helicase activities. Selected examples illustrate which conclusions can be drawn from the kinetic data obtained, highlight possible pitfalls in data analysis and interpretation, and outline how kinetic models can be related to functionally relevant states.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

Phytophthora effector PSR1 hijacks the host pre-mRNA splicing machinery to modulate small RNA biogenesis and plant immunity.

Phytophthora effector PSR1 suppresses small RNA (sRNA)-mediated immunity in plants, but the underlying mechanism remains unknown. Here, we show that Phytophthora suppressor of RNA silencing 1 (PSR1) contributes to the pathogenicity of Phytophthora sojae and specifically binds to three conserved C-terminal domains of the eukaryotic PSR1-Interacting Protein 1 (PINP1). PINP1 encodes PRP16, a core pre-mRNA splicing factor that unwinds RNA duplexes and binds to primary microRNA transcripts and general RNAs. Intriguingly, PSR1 decreased both RNA helicase and RNA-binding activity of PINP1, thereby dampening sRNA biogenesis and RNA metabolism. The PSR1-PINP1 interaction caused global changes in alternative splicing (AS). A total of 5,135 genes simultaneously exhibited mis-splicing in both PSR1-overexpressing and PINP1-silenced plants. AS upregulated many mRNA transcripts that had their introns retained. The high occurrence of intron retention in AS-induced transcripts significantly promoted Phytophthora pathogen infection in Nicotiana benthamiana, and this might be caused by the production of truncated proteins. Taken together, our findings reveal a key role for PINP1 in regulating sRNA biogenesis and plant immunity.

In vitro characterization of Dhr1 from Saccharomyces cerevisiae.

The RNA helicase Dhr1 from S. cerevisiae is an essential enzyme required for the assembly of the cytosolic small ribosomal subunit (SSU). A critical feature of the SSU is the central pseudoknot, an RNA fold that organizes the overall architecture of the subunit and connects all four domains of the 18S ribosomal RNA (rRNA). The initial folding of rRNA is guided, in part, by the U3 small nucleolar RNA, which base-pairs with the pre-rRNA in such a way as to preclude premature formation of the central pseudoknot. Thus, the essential role of Dhr1 is the unwinding of U3 from the pre-rRNA to allow folding of the central pseudoknot. Enzymes of the DEAH/RNA helicase A-like (RHA) family, to which Dhr1 belongs, are involved in splicing and ribosome biogenesis. They typically unwind RNA duplexes by translocation along a single strand of RNA in a 3' to 5' direction, driven by ATP hydrolysis. The substrate specificity of these enzymes requires tight regulation of their activity, by restricting access to their substrates, requiring adaptors to recruit them to their substrates and mechanisms of inhibiting and activating their activity. Purified Dhr1 is an active RNA-dependent ATPase with specific unwinding activity. Here, we provide detailed protocols for its purification and assays for its ATPase and unwinding activities.

Investigating the N‐ and C‐terminal Extensions of Yeast DEAD‐box Protein Dbp6

DEAD-box proteins are RNA-dependent ATPases that are involved in all aspects of RNA metabolism. Even though these enzymes are highly conserved in both sequence and structure, each DEAD-box protein has a unique intracellular function and is therefore essential. To gain insight into this enzymatic specificity, we are investigating the significance of the 192 amino acid N-terminal and 63 amino acid C-terminal extensions of Dbp6, a protein required for yeast ribosome assembly. To find the minimum functional length of the flanking region, serial truncation constructs were designed and their effect on the growth of yeast cells in which endogenous Dbp6 has been depleted was observed. The results indicate that deletion of up to 168 amino acids at the N-terminus (ΔN168) or 27 amino acids at the C-terminus (ΔC27) give either wild-type level or slow growth. However, removal of five more residues at the N-terminus or two more at the C-terminus results in a lethal phenotype. Interestingly, even though Dbp6 ΔN143 and Dbp6 ΔC21 each have near normal growth, the double truncation construct Dbp6 ΔN143ΔC21 has no growth. Western blot analysis supported the hypothesis that a non-functional protein was present. We are currently cloning the truncation constructs into bacterial expression vectors in order to over-express, purify, and test the enzymatic activity of the truncated proteins. Additionally, we are using site-directed mutagenesis to determine the effect of single amino acid mutations in the N-terminus and conserved core. Future experiments include measuring the ATP and RNA binding and RNA duplex unwinding activities of truncated Dbp6 in vitro. Understanding the enzymatic function of Dbp6 in vitro will elucidate its specific cellular roles and allow for further research on the involvement of Dbp6 and its human ortholog DDX51 in cell proliferation, viability, and cancer signaling. Gaining insight into the normal and impaired functions of DDX51 through this yeast model system will hopefully lead to future research on the potential therapeutic targeting of DEAD-box proteins in cancer treatment.

Regulation of RNA helicase activity: principles and examples.

RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for non-sequence-specific interaction with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

Modulation of Ago2 Loading by Cyclophilin 40 Endows a Unique Repertoire of Functional miRNAs during Sperm Maturation in Drosophila

In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. Here, we identify fly Cyclophilin 40 (Cyp40) as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC formation. We show that testis-distinctive Ago-sorting and strand-selection mechanisms accumulate a unique set of miRNAs on Ago2. Cyp40 interacts with duplex-incorporating Ago2 through Hsp90 invitro and selectively promotes the build-up of Ago2-bound miRNAs, but not endogenous siRNAs, invivo. Moreover, one of Cyp40-dependent Ago2-sorted miRNAs is required for late spermatogenesis, unraveling the physiological relevance of the unconventional yet conserved Drosophila miRNA-Ago2 sorting pathway. Collectively, these results identify RISC-regulatory roles for Hsp90 machinery and, more generally, highlight the tissue-specific adaptation of sRNA pathways through chaperone diversification.

Role reversal of functional identity in host factors: Dissecting features affecting pro-viral versus antiviral functions of cellular DEAD-box helicases in tombusvirus replication.

Positive-stranded (+)RNA viruses greatly exploit host cells to support viral replication. However, unlike many other pathogens, (+)RNA viruses code for only a limited number of genes, making them highly dependent on numerous co-opted host factors for supporting viral replication and other viral processes during their infections. This excessive dependence on subverted host factors, however, renders (+)RNA viruses vulnerable to host restriction factors that could block virus replication. Interestingly, cellular ATP-dependent DEAD-box RNA helicases could promote or inhibit the replication of Tomato bushy stunt virus (TBSV) replication. However, it is currently unknown what features make a particular DEAD-box helicase either pro-viral or antiviral. In this work, we succeeded in reversing the viral function of the antiviral DDX17-like RH30 DEAD-box helicase by converting it to a pro-viral helicase. We also turned the pro-viral DDX3-like RH20 helicase into an antiviral helicase through deletion of a unique N-terminal domain. We demonstrate that in the absence of the N-terminal domain, the core helicase domain becomes unhinged, showing altered specificity in unwinding viral RNA duplexes containing cis-acting replication elements. The discovery of the sequence plasticity of DEAD-box helicases that can alter recognition of different cis-acting RNA elements in the viral genome illustrates the evolutionary potential of RNA helicases in the arms race between viruses and their hosts, including key roles of RNA helicases in plant innate immunity. Overall, these findings open up the possibility to turn the pro-viral host factors into antiviral factors, thus increasing the potential antiviral arsenal of the host for the benefit of agriculture and health science.

The Sac10b homolog from Sulfolobus islandicus is an RNA chaperone.

Nucleic acid-binding proteins of the Sac10b family, also known as Alba, are widely distributed in Archaea. However, the physiological roles of these proteins have yet to be clarified. Here, we show that Sis10b, a member of the Sac10b family from the hyperthermophilic archaeon Sulfolobus islandicus, was active in RNA strand exchange, duplex RNA unwinding in vitro and RNA unfolding in a heterologous host cell. This protein exhibited temperature-dependent binding preference for ssRNA over dsRNA and was more efficient in RNA unwinding and RNA unfolding at elevated temperatures. Notably, alanine substitution of a highly conserved basic residue (K) at position 17 in Sis10b drastically reduced the ability of this protein to catalyse RNA strand exchange and RNA unwinding. Additionally, the preferential binding of Sis10b to ssRNA also depended on the presence of K17 or R17. Furthermore, normal growth was restored to a slow-growing Sis10b knockdown mutant by overproducing wild-type Sis10b but not by overproducing K17A in this mutant strain. Our results indicate that Sis10b is an RNA chaperone that likely functions most efficiently at temperatures optimal for the growth of S. islandicus, and K17 is essential for the chaperone activity of the protein.

RNA duplex recognition and unwinding by DEAD-box helicase DDX3X

RNA duplex recognition and unwinding by DEAD-box helicase DDX3X

A newly identified duplex RNA unwinding activity of archaeal RNase J depends on processive exoribonucleolysis coupled steric occlusion by its structural archaeal loops

ABSTRACT RNase J is a prokaryotic 5′-3′ exo/endoribonuclease that functions in mRNA decay and rRNA maturation. Here, we report a novel duplex unwinding activity of mpy-RNase J, an archaeal RNase J from Methanolobus psychrophilus, which enables it to degrade duplex RNAs with hairpins up to 40 bp when linking a 5′ single-stranded overhangs of ≥ 7 nt, corresponding to the RNA channel length. A 6-nt RNA-mpy-RNase J-S247A structure reveals the RNA-interacting residues and a steric barrier at the RNA channel entrance comprising two archaeal loops and two helices. Mutagenesis of the residues key to either exoribonucleolysis or RNA translocation diminished the duplex unwinding activity. Substitution of the residues in the steric barrier yielded stalled degradation intermediates at the duplex RNA regions. Thus, an exoribonucleolysis-driven and steric occlusion-based duplex unwinding mechanism was identified. The duplex unwinding activity confers mpy-RNase J the capability of degrading highly structured RNAs, including the bacterial REP RNA, and archaeal mRNAs, rRNAs, tRNAs, SRPs, RNase P and CD-box RNAs, providing an indicative of the potential key roles of mpy-RNase J in pleiotropic RNA metabolisms. Hydrolysis-coupled duplex unwinding activity was also detected in a bacterial RNase J, which may use a shared but slightly different unwinding mechanism from archaeal RNase Js, indicating that duplex unwinding is a common property of the prokaryotic RNase Js.

A high ATP concentration enhances the cooperative translocation of the SARS coronavirus helicase nsP13 in the unwinding of duplex RNA

Severe acute respiratory syndrome coronavirus nonstructural protein 13 (SCV nsP13), a superfamily 1 helicase, plays a central role in viral RNA replication through the unwinding of duplex RNA and DNA with a 5′ single-stranded tail in a 5′ to 3′ direction. Despite its putative role in viral RNA replication, nsP13 readily unwinds duplex DNA by cooperative translocation. Herein, nsP13 exhibited different characteristics in duplex RNA unwinding than that in duplex DNA. nsP13 showed very poor processivity on duplex RNA compared with that on duplex DNA. More importantly, nsP13 inefficiently unwinds duplex RNA by increasing the 5′-ss tail length. As the concentration of nsP13 increased, the amount of unwound duplex DNA increased and that of unwound duplex RNA decreased. The accumulation of duplex RNA/nsP13 complexes increased as the concentration of nsP13 increased. An increased ATP concentration in the unwinding of duplex RNA relieved the decrease in duplex RNA unwinding. Thus, nsP13 has a strong affinity for duplex RNA as a substrate for the unwinding reaction, which requires increased ATPs to processively unwind duplex RNA. Our results suggest that duplex RNA is a preferred substrate for the helicase activity of nsP13 than duplex DNA at high ATP concentrations.

Function of Auxiliary Domains of the DEAH/RHA Helicase DHX36 in RNA Remodeling

The DEAH/RHA helicase DHX36 has been linked to cellular RNA and DNA quadruplex structures and to AU-rich RNA elements. In vitro, DHX36 remodels DNA and RNA quadruplex structures and unwinds DNA duplexes in an ATP-dependent manner. DHX36 contains the superfamily 2 helicase core and several auxiliary domains that are conserved in orthologs of the enzyme. The role of these auxiliary domains for the enzymatic function of DHX36 is not well understood. Here, we combine structural and biochemical studies to define the function of three auxiliary domains that contact nucleic acid. We first report the crystal structure of mouse DHX36 bound to ADP. The structure reveals an overall architecture of mouse DHX36 that is similar to previously reported architectures of fly and bovine DHX36. In addition, our structure shows conformational changes that accompany stages of the ATP-binding and hydrolysis cycle. We then examine the roles of the DHX36-specific motif (DSM), the OB-fold, and a conserved β-hairpin (β-HP) in mouse DHX36 in the remodeling of RNA structures. We demonstrate and characterize RNA duplex unwinding for DHX36 and examine the remodeling of inter- and intramolecular RNA quadruplex structures. We find that the DSM not only functions as a quadruplex binding adaptor but also promotes the remodeling of RNA duplex and quadruplex structures. The OB-fold and the β-HP contribute to RNA binding. Both domains are also essential for remodeling RNA quadruplex and duplex structures. Our data reveal roles of auxiliary domains for multiple steps of the nucleic acid remodeling reactions.

The mechanism of RNA duplex recognition and unwinding by DEAD-box helicase DDX3X

DEAD-box helicases (DDXs) regulate RNA processing and metabolism by unwinding short double-stranded (ds) RNAs. Sharing a helicase core composed of two RecA-like domains (D1D2), DDXs function in an ATP-dependent, non-processive manner. As an attractive target for cancer and AIDS treatment, DDX3X and its orthologs are extensively studied, yielding a wealth of biochemical and biophysical data, including structures of apo-D1D2 and post-unwound D1D2:single-stranded RNA complex, and the structure of a D2:dsRNA complex that is thought to represent a pre-unwound state. However, the structure of a pre-unwound D1D2:dsRNA complex remains elusive, and thus, the mechanism of DDX action is not fully understood. Here, we describe the structure of a D1D2 core in complex with a 23-base pair dsRNA at pre-unwound state, revealing that two DDXs recognize a 2-turn dsRNA, each DDX mainly recognizes a single RNA strand, and conformational changes induced by ATP binding unwinds the RNA duplex in a cooperative manner.

Abstract 2698: eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma

Abstract Dysregulated messenger RNA (mRNA) translation drives the pathogenesis of multiple hematological malignancies. In lymphoma this includes the upregulation of key driver oncogenes and anti-apoptotic proteins (e.g., MYC, CCND1/3, BCL2 and MCL1) that contain a highly structured 5’-untranslated region (UTR) in their mRNA requiring enhanced eIF4A helicase activity for translation. eIF4A is a component of the eIF4F translation initiation complex and catalyzes the ATP-dependent unwinding of RNA duplexes and facilitates 43S ribosome scanning within the 5’-UTR. The activation of oncogenic signaling pathways, including RAS and PI3K, enhance eIF4A activity through phosphorylation of eIF4B, eIF4G and PDCD4 which facilitates formation of eIF4F and full activation of eIF4A. The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, promoting the translation of oncogenes with complex 5’-UTRs that are required for tumor cell proliferation, survival and metastasis. eFT226 is a potent and sequence selective eIF4A1 inhibitor that promotes eIF4A1 binding to specific 5’-UTR polypurine and/or G-quadraplex recognition motifs leading to a selective block in ribosome mRNA scanning. The sequence dependency of eFT226 translational inhibition was evaluated in cell-based reporter assays demonstrating &amp;gt;100-fold greater sensitivity for reporter constructs containing a polypurine motif in the 5’-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex with increased drug residence time between eFT226, eIF4A1 and RNA oligonucleotides containing polypurine motifs. The ability of eFT226 to inhibit MYC or MCL1 expression was found to be dependent on the presence of their respective 5’-UTR supporting a translational regulation mechanism dependent on recognition elements within the 5’-UTR. eFT226 shows potent anti-proliferative activity (GI50 &amp;lt; 15 nM) against a panel of B-cell lymphoma cell lines. Treatment with eFT226 leads to coordinated inhibition of MYC, CCND1/3, BCL2 or MCL1 protein expression resulting in significant anti-tumor activity. eFT226 has good pharmacokinetic properties and exhibits significant in vivo activity across a panel of diffuse large B cell lymphoma (DLBCL), and Burkitt lymphoma tumor models with ≤1 mg/kg/week IV administration. Further evaluation of predictive markers of sensitivity or resistance has shown that tumors with mTOR mediated activation of eIF4A are most sensitive to eFT226. In addition, tumors with PTEN mutations do not exhibit activated eIF4A and are generally resistant to induction of apoptosis by eFT226, resulting in reduced in vivo efficacy. The association of eFT226 activity with PI3K/mTOR pathway activation and mutational status provides a means to identify patient subsets during clinical development. Clinical trials in patients with lymphoma and other malignancies are planned. Citation Format: Peggy A. Thompson, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Eric Sung, Ana Parra, Jocelyn Staunton, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Justin T. Ernst, Siegfried H. Reich, Kevin R. Webster. eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2698.

Investigating the N‐ and C‐terminal Extensions of the Yeast DEAD‐box Protein Dbp6

DEAD‐box proteins are RNA‐dependent ATPases that are involved in all aspects of RNA metabolism. Also known as DEAD‐box helicases, these enzymes bind RNA and ATP and unwind short RNA duplexes via a highly conserved helicase core. N‐ and C‐terminal extensions that flank this core differ in both length and composition between DEAD‐box proteins and have been shown to contribute to RNA specificity and binding, thereby conferring the diverse cellular roles of DEAD‐box proteins. Here, we are investigating the 192 amino acid N‐terminal and 63 amino acid C‐terminal extensions of Dbp6, a yeast DEAD‐box protein that is required for 60S ribosome biogenesis. To find the minimum functional length of the flanking regions, we have systematically designed truncation constructs and observed their effects on the growth of yeast cells in which endogenous Dbp6 has been depleted. Our results indicate that deletion of up to 168 amino acids at the N‐terminus (ΔN168) or 27 amino acids at the C‐terminus (ΔC27) give either wild‐type level or slow growth. However, removal of five more residues at the N‐terminus or two more at the C‐terminus results in a lethal phenotype. Interestingly, even though Dbp6 ΔN143 and Dbp6 ΔC21 each have near normal growth, the double truncation construct Dbp6 ΔN143ΔC21 has no growth. We are currently conducting Western blot analysis to confirm that Dbp6 is indeed being expressed in these yeast cells with growth phenotypes. Follow‐up experiments include measuring the ATP and/or RNA binding and RNA duplex unwinding activities of Dbp6 truncation constructs in vitro. Since most of the yeast DEAD‐box proteins have orthologs in humans and many of those proteins have been implicated in cancer development or progression, we hope that these insights into the molecular workings of Dbp6 can contribute to our broader understanding of DEAD‐box proteins and their uses as potential cancer therapeutic targets.Support or Funding InformationEckerd College Natural Sciences Summer Research Program (NSSRP)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genome-Wide Discovery of DEAD-Box RNA Helicase Targets Reveals RNA Structural Remodeling in Transcription Termination

RNA helicases are a class of enzymes that unwind RNA duplexes in vitro but whose cellular functions are largely enigmatic. Here, we provide evidence that the DEAD-box protein Dbp2 remodels RNA-protein complex (RNP) structure to facilitate efficient termination of transcription in Saccharomyces cerevisiae via the Nrd1-Nab3-Sen1 (NNS) complex. First, we find that loss of DBP2 results in RNA polymerase II accumulation at the 3' ends of small nucleolar RNAs and a subset of mRNAs. In addition, Dbp2 associates with RNA sequence motifs and regions bound by Nrd1 and can promote its recruitment to NNS-targeted regions. Using Structure-seq, we find altered RNA/RNP structures in dbp2∆ cells that correlate with inefficient termination. We also show a positive correlation between the stability of structures in the 3' ends and a requirement for Dbp2 in termination. Taken together, these studies provide a role for RNA remodeling by Dbp2 and further suggests a mechanism whereby RNA structure is exploited for gene regulation.