CD spectra reveal the state of G-quadruplexes and i-motifs in repeated and other DNA sequences.

The B-DNA of the genome contains numerous sequences that can form various noncanonical structures including G-quadruplex (G4), formed by two or more stacks of four guanine residues in a plane, and intercalating motif (i-motif [iM]) formed by alternately arranged C-C pairs. One of the easy yet sensitive methods to study G4s and iMs is circular dichroism (CD) spectroscopy, which generates characteristic G4 and iM peaks. We have analyzed and compared the effects of various environmental factors including pH, buffer composition, temperature, flanking sequences, complimentary DNA strands, and single-stranded DNA binding protein (SSB) on the CD patterns of G4s and iMs generated by two groups of DNA molecules, one containing tandem repeats of GGGGCC and CCCCGG from the C9ORF72 gene associated with amyotrophic lateral sclerosis and frontotemporal dementia, and the second containing polyG/polyC clusters from oncogene promoter-proximal regions without such tandem repeats.

The RNA-binding protein PCBP1 modulates transcription by recruiting the G-quadruplex-specific helicase DHX9.

PCBP1, polycytosine (poly(C)) binding protein 1, an RNA and single-stranded DNA (ssDNA) binding protein, binds poly(C) DNA tracts but it remains unclear whether its ability to bind ssDNA contributes to transcriptional regulation. Here, we report that PCBP1's DNA binding sites are enriched at transcription start sites and that by binding to promoter regions, PCBP1 regulates transcription in addition to splicing and translation. At PCBP1 target genes, we show that PCBP1 interacts with several RNA/DNA hybrid (R-loop) associated G-quadruplex resolving helicases.

The ubiquitin E3 ligase ARIH1 regulates hnRNP E1 protein stability, EMT and breast cancer progression.

The epithelial to mesenchymal transition (EMT), a process that is aberrantly activated in cancer and facilitates metastasis to distant organs, requires coordinated transcriptional and post-transcriptional control of gene expression. The tumor-suppressive RNA binding protein, hnRNP-E1, regulates splicing and translation of EMT-associated transcripts and it is thought that it plays a major role in the control of epithelial cell plasticity during cancer progression. We have utilized yeast 2 hybrid screening to identify novel hnRNP-E1 interactors that play a role in regulating hnRNP-E1; this approach led to the identification of the E3 ubiquitin ligase ARIH1.

Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity.

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner.

Yeast Sphingolipid Phospholipase Gene Regulates the Spindle Checkpoint by a -Dependent Mechanism.

Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated.

TGFβ promotes breast cancer stem cell self-renewal through an ILEI/LIFR signaling axis.

FAM3C/Interleukin-like EMT Inducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFβ signaling pathway by 3'-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFβ stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates with enhanced invasion and migration.

Interleukin-like EMT inducer (ILEI) promotes melanoma invasiveness and is transcriptionally up-regulated by upstream stimulatory factor-1 (USF-1).

Interleukin-like EMT inducer (ILEI, ) is a secreted factor that contributes to the epithelial-to-mesenchymal transition (EMT), a cell-biological process that confers metastatic properties to a tumor cell. However, very little is known about how ILEI is regulated. Here we demonstrate that ILEI is an regulator of melanoma invasiveness and is transcriptionally up-regulated by the upstream stimulatory factor-1 (USF-1), an E-box-binding, basic-helix-loop-helix family transcription factor.

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest.

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1-Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression.

Identification and characterization of an hnRNP E1 translational silencing motif.

Non-canonical transforming growth factor β (TGFβ) signaling through protein kinase B (Akt2) induces phosphorylation of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) at serine-43 (p-hnRNP E1). This post-translational modification (PTM) of hnRNP E1 promotes its dissociation from a 3' untranslated region (UTR) nucleic acid regulatory motif, driving epithelial to mesenchymal transition (EMT) and metastasis. We have identified an hnRNP E1 consensus-binding motif and genomically resolved a subset of genes in which it is contained.

Mechanism of Regulation of Intrachromatid Recombination and Long-Range Chromosome Interactions in Saccharomyces cerevisiae.

The NAD-dependent histone deacetylase Sir2 controls ribosomal DNA (rDNA) silencing by inhibiting recombination and RNA polymerase II-catalyzed transcription in the rDNA of Saccharomyces cerevisiae Sir2 is recruited to nontranscribed spacer 1 (NTS1) of the rDNA array by interaction between the RENT ( RE: gulation of N: ucleolar S: ilencing and T: elophase exit) complex and the replication terminator protein Fob1. The latter binds to its cognate sites, called replication termini (Ter) or replication fork barriers (RFB), that are located in each copy of NTS1. This work provides new mechanistic insights into the regulation of rDNA silencing and intrachromatid recombination by showing that Sir2 recruitment is stringently regulated by Fob1 phosphorylation at specific sites in its C-terminal domain (C-Fob1), which also regulates long-range Ter-Ter interactions.

Computational Identification of Post Translational Modification Regulated RNA Binding Protein Motifs.

RNA and its associated RNA binding proteins (RBPs) mitigate a diverse array of cellular functions and phenotypes. The interactions between RNA and RBPs are implicated in many roles of biochemical processing by the cell such as localization, protein translation, and RNA stability. Recent discoveries of novel mechanisms that are of significant evolutionary advantage between RBPs and RNA include the interaction of the RBP with the 3' and 5' untranslated region (UTR) of target mRNA.

Finding pathway-modulating genes from a novel Ontology Fingerprint-derived gene network.

To enhance our knowledge regarding biological pathway regulation, we took an integrated approach, using the biomedical literature, ontologies, network analyses and experimental investigation to infer novel genes that could modulate biological pathways. We first constructed a novel gene network via a pairwise comparison of all yeast genes' Ontology Fingerprints--a set of Gene Ontology terms overrepresented in the PubMed abstracts linked to a gene along with those terms' corresponding enrichment P-values. The network was further refined using a Bayesian hierarchical model to identify novel genes that could potentially influence the pathway activities.

Identification of C18:1-phytoceramide as the candidate lipid mediator for hydroxyurea resistance in yeast.

Recent studies showed that deletion of ISC1, the yeast homologue of the mammalian neutral sphingomyelinase, resulted in an increased sensitivity to hydroxyurea (HU). This raised an intriguing question as to whether sphingolipids are involved in pathways initiated by HU. In this study, we show that HU treatment led to a significant increase in Isc1 activity.

Nicotinamide induces Fob1-dependent plasmid integration into chromosome XII in Saccharomyces cerevisiae.

In the ribosomal DNA (rDNA) array of Saccharomyces cerevisiae, DNA replication is arrested by the Fob1 protein in a site-specific manner that stimulates homologous recombination. The silent information regulator Sir2, which is loaded at the replication arrest sites by Fob1, suppresses this recombination event. A plasmid containing Fob1-binding sites, when propagated in a yeast strain lacking SIR2 is integrated into the yeast chromosome in a FOB1-dependent manner.

Hydroxyurea treatment inhibits proliferation of Cryptococcus neoformans in mice.

The fungal pathogen Cryptococcus neoformans (Cn) is a serious threat to immunocompromised individuals, especially for HIV patients who develop meningoencephalitis. For effective cryptococcal treatment, novel antifungal drugs or innovative combination therapies are needed. Recently, sphingolipids have emerged as important bioactive molecules in the regulation of microbial pathogenesis.

Cellular morphogenesis under stress is influenced by the sphingolipid pathway gene ISC1 and DNA integrity checkpoint genes in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, replication stress induced by hydroxyurea (HU) and methyl methanesulfonate (MMS) activates DNA integrity checkpoints; in checkpoint-defective yeast strains, HU treatment also induces morphological aberrations. We find that the sphingolipid pathway gene ISC1, the product of which catalyzes the generation of bioactive ceramides from complex sphingolipids, plays a novel role in determining cellular morphology following HU/MMS treatment. HU-treated isc1Δ cells display morphological aberrations, cell-wall defects, and defects in actin depolymerization.

The intra-S phase checkpoint protein Tof1 collaborates with the helicase Rrm3 and the F-box protein Dia2 to maintain genome stability in Saccharomyces cerevisiae.

The intra-S phase checkpoint protein complex Tof1/Csm3 of Saccharomyces cerevisiae antagonizes Rrm3 helicase to modulate replication fork arrest not only at the replication termini of rDNA but also at strong nonhistone protein binding sites throughout the genome. We investigated whether these checkpoint proteins acted either antagonistically or synergistically with Rrm3 in mediating other important functions such as maintenance of genome stability. High retromobility of a normally quiescent retrovirus-like transposable element Ty1 of S.

Replication fork arrest and rDNA silencing are two independent and separable functions of the replication terminator protein Fob1 of Saccharomyces cerevisiae.

The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other as well as functionally separable.

Contrasting roles of checkpoint proteins as recombination modulators at Fob1-Ter complexes with or without fork arrest.

The replication terminator protein Fob1 of Saccharomyces cerevisiae specifically interacts with two tandem Ter sites (replication fork barriers) located in the nontranscribed spacer of ribosomal DNA (rDNA) to cause polar fork arrest. The Fob1-Ter complex is multifunctional and controls other DNA transactions such as recombination by multiple mechanisms. Here, we report on the regulatory roles of the checkpoint proteins in the initiation and progression of recombination at Fob1-Ter complexes.

Hydroxyurea sensitivity reveals a role for ISC1 in the regulation of G2/M.

Saccharomyces cerevisiae cells lacking ISC1 (inositol phosphosphingolipase C) exhibit sensitivity to genotoxic agents such as methyl methanesulfonate and hydroxyurea (HU). Cell cycle analysis by flow cytometry revealed a G(2)/M block in isc1Delta cells when treated with methyl methanesulfonate or HU. Further investigation revealed that the levels of Cdc28 phosphorylated on Tyr-19, which plays an essential role in the regulation of the G(2)/M checkpoint, were higher in synchronized and asynchronous cells lacking ISC1 in response to HU.

The Tof1p-Csm3p protein complex counteracts the Rrm3p helicase to control replication termination of Saccharomyces cerevisiae.

Termination of replication forks at the natural termini of the rDNA of Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by the interaction of the Fob1p replication terminator protein with the tandem Ter sites located in the nontranscribed spacers. Here we show, by both 2D gel analyses and chromatin immunoprecipitations (ChIP), that there exists a second level of global control mediated by the intra-S-phase checkpoint protein complex of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity of the Rrm3p helicase ("sweepase"). The sweepase tends to release arrested forks presumably by the transient displacement of the Ter-bound Fob1p.

Binding of the replication terminator protein Fob1p to the Ter sites of yeast causes polar fork arrest.

Fob1p protein has been implicated in the termination of replication forks at the two tandem termini present in the non-transcribed spacer region located between the sequences encoding the 35 S and the 5 S RNAs of Saccharomyces cerevisiae. However, the biochemistry and mode of action of this protein were previously unknown. We have purified the Fob1p protein to near-homogeneity, and we developed a novel technique to show that it binds specifically to the Ter1 and Ter2 sequences.