Lipid biosynthesis perturbation impairs endoplasmic reticulum-associated degradation.

The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1-Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae. This screen revealed that INO4 is required for efficient Deg1-Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1-Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.

Differential Gene Expression following DHX36/G4R1 Knockout Is Associated with G-Quadruplex Content and Cancer.

G-quadruplexes (G4s) are secondary DNA and RNA structures stabilized by positive cations in a central channel formed by stacked tetrads of Hoogsteen base-paired guanines. G4s form from G-rich sequences across the genome, whose biased distribution in regulatory regions points towards a gene-regulatory role. G4s can themselves be regulated by helicases, such as DHX36 (aliases: G4R1 and RHAU), which possess the necessary activity to resolve these stable structures. G4s have been shown to both positively and negatively regulate gene expression when stabilized by ligands, or through the loss of helicase activity. Using DHX36 knockout Jurkat cell lines, we identified widespread, although often subtle, effects on gene expression that are associated with the presence or number of observed G-quadruplexes in promoters or gene regions. Genes that significantly change their expression, particularly those that show a significant increase in RNA abundance under DHX36 knockout, are associated with a range of cellular functions and processes, including numerous transcription factors and oncogenes, and are linked to several cancers. Our work highlights the direct and indirect role of DHX36 in the transcriptome of T-lymphocyte leukemia cells and the potential for DHX36 dysregulation in cancer.

The RNA helicase DHX36-G4R1 modulates C9orf72 GGGGCC hexanucleotide repeat-associated translation.

GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36-G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length-dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome.

The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3-R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5' capped guanosine (Cap1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines (Cap2G or Cap3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer-dimer equilibrium properties of Cap1G, Cap2G, and Cap3G 5'-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the Cap1G 5'-leader RNA adopts a dimeric structure, the Cap2G and Cap3G 5'-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimer-promoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5' guanosine.

Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1.

Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.

G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.

It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent K(d)'s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these K(d)'s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structures.

Macro-ER-phagy receptors Atg39p and Atg40p confer resistance to aminoglycoside hygromycin B in S. cerevisiae.

Receptor-mediated autophagic turnover of portions of the endoplasmic reticulum (ER) is mediated by macro-ER-phagy. We hypothesized macro-ER-phagy promotes proteotoxic stress resistance. We predicted Saccharomyces cerevisiae lacking macro-ER-phagy receptors would exhibit enhanced sensitivity to hygromycin B, which reduces translational fidelity and is expected to globally disrupt protein homeostasis, including at the ER. We observed that loss of either of two yeast macro-ER-phagy receptors (Atg39p or Atg40p) compromised cellular resistance to hygromycin B to a similar extent as loss of ER-associated degradation (ERAD) ubiquitin ligases Hrd1p and Doa10p. Our data are consistent with a model whereby macro-ER-phagy and ERAD collaborate to mediate ER protein quality control. Disruptions of macro-ER-phagy have been linked to neuropathy, dementia, and cancer. A dampened capacity to mediate protein quality control may contribute to these conditions.

"Important enough to show the world": Using Authentic Research Opportunities and Micropublications to Build Students' Science Identities.

Primarily undergraduate institutions (PUIs) often struggle to provide authentic research opportunities that culminate in peer-reviewed publications due to "recipe-driven" lab courses and the comprehensive body of work necessary for traditional scientific publication. However, the advent of short-form, single-figure "micropublications" has created novel opportunities for early-career scientists to make and publish authentic scientific contributions on a scale and in a timespan compatible with their training periods. The purpose of this qualitative case study is to explore the benefits accrued by eight undergraduate and master's students who participated in authentic, small-scale research projects and disseminated their work as coauthors of peer-reviewed micropublications at a PUI. In these interviews, students reported that through the process of conducting and publishing their research, they developed specific competencies: reading scientific literature, proposing experiments, and collecting/interpreting publication-worthy data. Further, they reported this process enabled them to identify as contributing members of the greater scientific community.

Inner Nuclear Membrane Asi Ubiquitin Ligase Catalytic Subunits Asi1p and Asi3p, but not Asi2p, confer resistance to aminoglycoside hygromycin B in Saccharomyces cerevisiae.

The heterotrimeric Asi ubiquitin ligase (encoded by ASI1, ASI2, and ASI3) mediates protein degradation in the inner nuclear membrane in Saccharomyces cerevisiae. Asi1p and Asi3p possess catalytic domains, while Asi2p functions as an adaptor for a subset of Asi substrates. We hypothesized the Asi complex is an important mediator of protein quality control, and we predicted that Asi would be required for optimal growth in conditions associated with elevated abundance of aberrant proteins. Loss of Asi1p or Asi3p, but not Asi2p, sensitized yeast to hygromycin B, which promotes translational infidelity by distorting the ribosome A site. Surprisingly, loss of quality control ubiquitin ligase Hul5p did not sensitize yeast to hygromycin B. Our results are consistent with a prominent role for an Asi subcomplex that includes Asi1p and Asi3p (but not Asi2p) in protein quality control.

Loss of protein quality control gene UBR1 sensitizes Saccharomyces cerevisiae to the aminoglycoside hygromycin B.

Ubr1 is a conserved ubiquitin ligase involved in the degradation of aberrant proteins in eukaryotic cells. The human enzyme is found mutated in patients with Johanson-Blizzard syndrome. We hypothesized that Ubr1 is necessary for optimal cellular fitness in conditions associated with elevated abundance of aberrant and misfolded proteins. Indeed, we found that loss of Ubr1 in the model eukaryotic microorganism Saccharomyces cerevisiae strongly sensitizes cells to hygromycin B, which reduces translational fidelity by causing ribosome A site distortion. Our results are consistent with a prominent role for Ubr1 in protein quality control. We speculate that disease manifestations in patients with Johanson-Blizzard syndrome are linked, at least in part, to defects in protein quality control caused by loss of Ubr1 function.

G-Quadruplex Helicase DHX36/G4R1 Engages Nuclear Lamina Proteins in Quiescent Breast Cancer Cells.

G-quadruplexes (G4s) are nucleic acid structures found enriched within gene regulatory sequences. G4s control fundamental cellular processes, including replication, transcription, and translation. Proto-oncogenes are enriched with G4 sequences, while tumor-suppressor genes are depleted, suggesting roles for G4s in cell survival and proliferation. Specialized helicases participate in G4-mediated gene regulation via enzymatic unwinding activity. One such enzyme, DHX36/G4R1, is the major G4-helicase and is a master regulator of G4-DNAs and mRNAs. G4-resolution promotes the expression of proproliferative genes; as such, DHX36/G4R1 promotes cell proliferation. Little is known about how DHX36/G4R1 itself is regulated in nondividing cells. We hypothesized that DHX36/G4R1 protein binding partners are altered when a cell transitions from a dividing to a quiescent state. We found that DHX36/G4R1 co-purifies with a distinct set of proteins under quiescent conditions, which may represent a novel complex that regulates DHX36/G4R1 during cell cycle transitions and have implications for development and cancer.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are >375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

Aggregation of Mod5 is affected by tRNA binding with implications for tRNA gene-mediated silencing.

Mod5 is a multifunctional protein that modifies a subset of tRNAs in the cytoplasm and is also required for an RNA-mediated form of transcriptional silencing. Previous in vivo studies have shown that the nuclear silencing function of Mod5 does not require that the causative tRNA gene encode a Mod5 substrate, although Mod5 is still required. However, previous data have not directly tested whether Mod5 can directly bind substrate and nonsubstrate RNAs. We herein demonstrate that Mod5 directly binds to both substrate and nonsubstrate RNAs, including a highly structured, non-tRNA sequence (5S-rRNA), consistent with previous in vivo data. Furthermore, we show that some RNAs drastically change the aggregation behavior of Mod5 with implications for tRNA gene-mediated silencing.

A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3'-Tail Sequences Are Sufficient for Tight and Complete Binding.

Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent Kd. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K+-induced "3+1" G4-topology with an apparent Kd = 10 ± 1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na+-induced "2+2" basket G4-structure formed by the same DNA sequence with an apparent Kd = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5' G4-structure with a 3' unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5' quadruplex with an opened 3'tail structure that is recognized by G4R1.

Human papillomavirus and tobacco use in tongue base cancers.

Human papillomavirus 16 (HPV-16) infection and tobacco use are associated with human oropharyngeal cancers. We conducted a study of the role of HPV and tobacco use in base of the tongue (BOT) cancers. DNA from 34 such cancers was subjected to HPV-16 and HPV-18-specific polymerase chain reaction analysis. Demographic and clinicopathologic data were obtained from each patient's medical record. HPV-16 was detected in 68% of tumors. Tobacco use was the only factor found to be significantly associated with HPV status. Tumors from 100% of patients who had never used tobacco tested positive for HPV, compared with only 56% of those who had ever used tobacco (Fisher exact test, p = 0.024). All tumors were associated with either tobacco use or HPV infection. These findings are consistent with the hypothesis that either tobacco use or HPV infection is necessary to the etiology of BOT tumors, and they suggest that tongue base carcinoma may be prevented by combining HPV vaccination with tobacco avoidance.

Chronic periodontitis-human papillomavirus synergy in base of tongue cancers.

OBJECTIVE: To assess whether chronic periodontitis history predicts human papillomavirus (HPV) status in patients with base of tongue cancers. DESIGN: Case-control study using existing patient data. SETTING: Roswell Park Cancer Institute. PATIENTS: Thirty patients newly diagnosed with base of tongue squamous cell carcinoma between 1999 and 2005 for whom both tumor samples and periodontal records were available. Patients younger than 21 years, edentulous, immunocompromised, and those with a history of cancer were excluded. Periodontitis history was assessed on the basis of alveolar bone loss (in millimeters) from panoramic radiographs by one examiner who was blinded to cancer status. MAIN OUTCOME MEASURE: HPV-16 and HPV-18 DNA were identified on paraffin-embedded tumor samples by polymerase chain reaction. Multiple logistic regression was used to estimate odds ratios and 95% confidence intervals. RESULTS: The prevalence of tumors positive for HPV-16 DNA was 21 of 30 (70%). None of the samples were positive for HPV-18 DNA. Compared with participants with HPV-negative tumors, patients with HPV-positive tumors had significantly higher mean alveolar bone loss (3.90 mm vs 2.85 mm, P = .01). After adjustment for age at diagnosis, sex, race/ethnicity, alcohol use, smoking status, and number of missing teeth, every millimeter of alveolar bone loss was associated with an approximately 4-fold (odds ratio, 3.96; 95% confidence interval, 1.18-13.36) increased risk of HPV-positive tumor status. Number of missing teeth was not associated with tumor HPV status (odds ratio, 0.95; 95% confidence interval, 0.74-1.21). CONCLUSIONS: Chronic periodontitis may be a significant factor in the natural history of HPV infection in patients with base of tongue cancers. Additional confirmation in larger studies is required.