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RNA biology is revolutionized by recent developments of diverse high-throughput technologies for transcriptome-wide profiling of molecular RNA structures. RNA structurome profiling data can be used to identify differentially structured regions between groups of samples. Existing methods are limited in scope to specific technologies and/or do not account for biological variation. Here, we present dStruct which is the first broadly applicable method for differential analysis accounting for biological variation in structurome profiling data. dStruct is compatible with diverse profiling technologies, is validated with experimental data and simulations, and outperforms existing methods.

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.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.05.027 Byline: Elizabeth J. Tran (1), Susan R. Wente (1) Abstract: The exchange of molecules between the nucleus and cytoplasm is mediated through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Altering the interactions between transport receptors and their cargo has been shown to be a major regulatory mechanism to control traffic through NPCs. New evidence now suggests that NPC proteins play active roles in translocation, and that transport is also controlled by dynamic changes in NPC composition and architecture. This view of ever-changing NPCs necessitates the re-evaluation of current models of nuclear transport and how this process is regulated. Author Affiliation: (1) Department of Cell and Developmental Biology, Vanderbilt University Medical Center, U-3209 MRBIII, 465 21.sup.st Avenue South, Nashville, TN 37232 USA

G-quadruplexes (G4) are noncanonical secondary structures formed in guanine-rich DNA and RNA sequences. MYC, one of the most critical oncogenes, forms a DNA G4 in its proximal promoter region (MycG4) that functions as a transcriptional silencer. However, MycG4 is highly stable in vitro and its regulatory role would require active unfolding. Here we report that DDX5, one of the founding members of the DEAD-box RNA helicase family, is extremely proficient at unfolding MycG4-DNA. Our results show that DDX5 is a highly active G4-resolvase that does not require a singlestranded overhang and that ATP hydrolysis is not directly coupled to G4-unfolding of DDX5. The chromatin binding sites of DDX5 are G-rich sequences. In cancer cells, DDX5 is enriched at the MYC promoter and activates MYC transcription. The DDX5 interaction with the MYC promoter and DDX5-mediated MYC activation is inhibited by G4-interactive small molecules. Our results uncover a function of DDX5 in resolving DNA and RNA G4s and suggest a molecular target to suppress MYC for cancer intervention. [ABSTRACT FROM AUTHOR]

Haloferax volcanii pre-tRNATrp processing requires box C/D ribonucleoprotein (RNP)-guided 2'-O-methylation of nucleotides C34 and U39 followed by intron excision. Positioning of the box C/D guide RNA within the intron of this pre-tRNA led to the assumption that nucleotide methylation is guided by the cis-positioned box C/D RNPs. We have now investigated the mechanism of 2'-O-methylation for the H. volcanii pre-tRNATrp in vitro by assembling methylation-competent box C/D RNPs on both the pre-tRNA and the excised intron (both linear and circular forms) using Methanocaldococcus jannaschii box C/D RNP core proteins. With both kinetic studies and single nucleotide substitutions of target and guide nucleotides, we now demonstrate that pre-tRNA methylation is guided in trans by the intron-encoded box C/D RNPs positioned in either another pre-tRNATrp or in the excised intron. Methylation by in vitro assembled RNPs prefers but does not absolutely require Watson. Crick pairing between the guide and target nucleotides. We also demonstrate for the first time that methylation of two nucleotides guided by a single box C/D RNA is sequential, that is, box C'/D' RNP-guided U39 methylation first requires box C/D RNP-guided methylation of C34. Methylation of the two nucleotides of exogenous pre-tRNATrp added to an H. volcanii cell extract also occurs sequentially and is also accomplished in trans using RNPs that pre-exist in the extract. Thus, this trans mechanism is analogous to eukaryal pre-rRNA 2'-O-methylation guided by intronencoded but trans-acting box C/D small nucleolar RNPs. This trans mechanism could explain the observed accumulation of the excised H. volcanii pretRNATrp intron in vivo. A trans mechanism would also eliminate the obligatory refolding of the pre-tRNA that would be required to carry out two cis-methylation reactions before pre-tRNA splicing. [ABSTRACT FROM AUTHOR]

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 dbp2Δ 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. [ABSTRACT FROM AUTHOR]

Cells fine-tune their metabolic programs according to nutrient availability in order to maintain homeostasis. This is achieved largely through integrating signaling pathways and the gene expression program, allowing cells to adapt to nutritional change. Dbp2, a member of the DEAD-box RNA helicase family in Saccharomyces cerevisiae, has been proposed to integrate gene expression with cellular metabolism. Prior work from our laboratory has reported the necessity of DBP2 in proper gene expression, particularly for genes involved in glucose-dependent regulation. Here, by comparing differentially expressed genes in dbp2Δ to those of 700 other deletion strains from other studies, we find that CYC8 and TUP1, which form a complex and inhibit transcription of numerous genes, corepress a common set of genes with DBP2. Gene ontology (GO) annotations reveal that these corepressed genes are related to cellular metabolism, including respiration, gluconeogenesis, and alternative carbon-source utilization genes. Consistent with a direct role in metabolic gene regulation, loss of either DBP2 or CYC8 results in increased cellular respiration rates. Furthermore, we find that corepressed genes have a propensity to be associated with overlapping long noncoding RNAs and that upregulation of these genes in the absence of DBP2 correlates with decreased binding of Cyc8 to these gene promoters. Taken together, this suggests that Dbp2 integrates nutrient availability with energy homeostasis by maintaining repression of glucose-repressed, Cyc8-targeted genes across the genome. [ABSTRACT FROM AUTHOR]

Cellular homeostasis requires a fine balance between energy uptake, utilization, and growth. Dbp2 is a member of the DEAD-box protein family in Saccharomyces cerevisiae with characterized ATPase and helicase activity in vitro. DEAD-box RNA helicases are a class of enzymes that utilize ATP hydrolysis to remodel RNA and/or RNA-protein (RNP) composition. Dbp2 has been proposed to utilize its helicase activity in vivo to promote RNA-protein complex assembly of both messenger (m)RNAs and long noncoding (lnc)RNAs. Previous work from our laboratory demonstrated that loss of DBP2 enhances the lncRNA-dependent transcriptional induction of the GAL genes by abolishing glucose-dependent repression. Herein, we report that either a carbon source switch or glucose deprivation results in rapid export of Dbp2 to the cytoplasm. Genome-wide RNA sequencing identified a new class of antisense hexose transporter transcripts that are specifically upregulated upon loss of DBP2. Further investigation revealed that both sense and antisense hexose transporter (HXT) transcripts are aberrantly expressed in DBP2-deficient cells and that this expression pathway can be partially mimicked in wild-type cells by glucose depletion. We also find that Dbp2 promotes ribosome biogenesis and represses alternative ATP-producing pathways, as loss of DBP2 alters the transcript levels of ribosome biosynthesis (snoRNAs and associated proteins) and respiration gene products. This suggests that Dbp2 is a key integrator of nutritional status and gene expression programs required for energy homeostasis. [ABSTRACT FROM AUTHOR]

Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genome-wide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Long non-coding RNAs (lncRNAs) are a group of molecules that function in gene regulation in yeast, plants and mammals. The precise mechanisms of action for lncRNAs, however, remain largely unclear. The GAL gene cluster has been used as a model system to study the function of these molecules in Saccharomyces cerevisiae, with a historical focus on lncRNA-dependent repression. Strikingly, in characterizing the role of the RNA helicase Dbp2, we discovered that the GAL lncRNAs could also promote transcriptional activation of the targeted GAL protein-coding genes. Interestingly, these lncRNAs help determine how quickly the GAL genes can be induced in response to galactose, without altering final steady-state transcript levels. This unexpected finding suggests that one role for lncRNAs is to promote the timing of gene expression. Herein, we discuss our discoveries in the context of current models of lncRNA functions in eukaryotes, with a key emphasis on future challenges for genomic research.