MOLECULAR, CELLULAR AND DEVELOPMENTAL BIOLOGY
Analysis of Lamin A and Progerin Biogenesis Using Nucleus Localization Signal Deleted Mutants
Presenter: Di Wu Status: Graduate Student
Authors: DI WU, ANDREW FLANNERY AND KAN CAO
Abstract: Lamin A is a major component of the nuclear lamina, which creates a dynamic network underneath the nuclear envelope, providing important mechanical supports. Mutations in the lamin A gene (LMNA) can cause severe genetic disorders, one of which is Hutchinson-Gilford progeria syndrome (HGPS) triggered by a dominant lamin A mutant named progerin. Unlike wild-type lamin A, whose c-terminal farnesyl group is excised to liberate it from the nuclear membrane, progerin maintains the farnesyl tail and accumulates on the nuclear membrane, leading to abnormal nuclear morphology during interphase and nuclear disassembly and reassembly defects during mitosis. Notably, progerin forms cytoplasmic aggregates after mitosis, which may potentially interfere with the normal cytoplasmic functions. To examine the effects of cytoplasmic progerin, nuclear localization signal (NLS)-deleted progerin and lamin A (PGΔNLS and LAΔNLS, respectively) were constructed and expressed as fusion proteins with EGFP reporter. Both ΔNLS mutants were farnesylated and restrained within the ER by their farnesyl tails. Blocking farnesylation with farnesyltransferase inhibitors (FTIs) promotes the release of LAΔNLS from the ER membranes, resembling the effects of non-farnesylable SSIMΔNLS mutants. Moreover, both ΔNLS mutants disturb normal emerin nuclear localization, and PGΔNLS severely affects ER disassembly at the onset of mitosis. These results reveal novel insights into lamin A biogenesis and the cytoplasmic effects of progerin.
Arabidopsis TSO1-MYB3R1 cell cycle regulatory modulecoordinates cell proliferation with differentiation at the plant stem cell niche
Presenter: Wanpeng Wang Status: Graduate Student
Authors: Wanpeng Wang and Zhongchi Liu
Abstract: Establishing functional organs during the development of multicellular organisms requires coordinated cell proliferation and differentiation. The formation of flowers, and hence plants’ ability to reproduce, depends on proper regulation of pluripotent stem cells at the shoot apical meristem (SAM). Previous studies discovered regulatory feedback circuitries that maintain stem cell homeostasis at the shoot and root meristems. However, how the cell cycle regulation is integrated with these feedback circuitries is not well understood. The Arabidopsis TSO1 gene plays an important role in meristem homeostasis and floral organ differentiation. tso1 mutants have significantly enlarged SAM with many more floral buds, yet these floral buds fail to differentiate into fertile floral organs.TSO1 encodes a putative transcription factor with two cysteine-rich CXC domains, which bind DNA. TSO1 homologs were found in animals, and they are components of the master cell cycle regulatory protein complex, the dREAM complex. Mutations in the dREAM complex subunits often lead to cancer and developmental defects, especially in the germ line of animals. We hypothesize that a dREAM-like complex may exist in plant, of which TSO1 is a key component. Through an EMS mutagenesis, we isolated 45 genetic suppressors of tso1-1. Three suppressor mutations were mapped to the MYB3R1 locus through “mapping-by-sequencing”. Additional 19 suppressors were also assigned to the MYB3R1 locus based on complementation tests and sequencing. The suppression of tso1 mutant phenotype by myb3r1 was confirmed by CRISPR genome editing that knocked out MYB3R1 in different tso1 alleles. Hence, MYB3R1 must act in the same process as TSO1 to regulate stem cell homeostasis at SAM. The Arabidopsis MYB3R1 is a close homolog of human oncogene Myb, which was previously shown to function in the animal dREAM complex that regulates cell cycle gene expression. We further showed that MYB3R1 is mis- and over-expressed in tso1 mutants. Our data suggest that a dREAM-like complex inArabidopsis, consisting of TSO1 and MYB3R1, may negatively regulate MYB3R1 and other cell cycle genes at SAM to coordinate cell proliferation with differentiation. Our finding provides important new insights into the mechanism of cell cycle regulation at stem cell niche of plants and has broad implication on animal stem cell regulation and cancer.
Auxin regulates nitrogen remobilization from poplar bark
Presenter: Gen Li Status: Graduate Student
Authors: Gen Li, Rongshuang Lin, Chioma Egekwu, Angus Murphy, Wendy Peer, Gary Coleman
Abstract: Abstract Although both annual and perennial plants cycle nitrogen (N) internally throughout the growing season, only perennial plants recycle N on a seasonal basis. The seasonal cycling of N by perennial plants is a major determinant of their lifestyle. In Populus, the bark storage proteins (BSP) class of vegetative storage proteins are central to seasonal N cycling. When BSP is catabolized in the spring, the released N is remobilized to meet the N demand of newly growing tissues, such as expanding buds and young leaves and significantly contributes to new growth. However, the mechanisms that initiate and regulate BSP catabolism remain unclear. In this study, we analyzed gene expression using DNA microarrays to profile genome-wide changes in gene expression in poplar bark during shoot regrowth following dormancy. Our data showed an increase in bark expression of both auxin transporters and auxin responsive genes (ARF and IAA) during BSP catabolism and shoot regrowth, which also coincided with the increased expression of specific protease genes. Furthermore, the expression of members of two important gene families in auxin-biosynthesis, YUCCAs and TAA1/TARs, were also induced in buds during shoot regrowth. The role of bud and/or shoot produced auxin in bark N remobilization was confirmed by blocking stem auxin transport using the NPA resulting in repression of BSP degradation. This repression was associated with significant decreases in bark expression of auxin transporters and IAA genes. Moreover, the bark expression of specific proteases was also down-regulated when auxin transport was blocked with NPA and expression of these same protease genes were induced with IAA treatment. Thus expanding buds and shoots may serve as the auxin source during bark N remobilization. These results indicate a critical role for auxin in modulating N remobilization during shoot regrowth by regulating expression of specific proteases.
Determining the Path of Auxin During Open-Air Phototropism in Arabidopsis Seedlings
Presenter: Candace Pritchard Status: Graduate Student
Authors: Candace Pritchard, Guojie Ma, Gregory Richter, John Christie, and Angus Murphy
Abstract: Despite more than a century of study, the process of phototropism, or plant growth toward a unilateral light source, is still not fully understood. During phototropism unilateral blue light is perceived via phot1, which then phosphorylates and inactivates ABCB19, resulting in a halt in vertical growth and pooling of auxin in the upper hypocotyl. Visualization of the DII:VENUS auxin reporter indicates that asymmetric auxin distributions on the lit and shaded sides of the hypocotyl are generated, though how this is achieved is unclear. Later events involve the transport of auxin down the shaded side of the hypocotyl to the elongation zone, resulting in bending towards the light source. Traditionally dark-grown seedlings are used to study this phenomenon, however, this approach fails to separate phototropism and photomorphogenesis, which then occur simultaneously upon light irradiation. Additionally, growth on vertical media during these assays makes it impossible to account for the impacts of media interaction during phototropism. Here, blue light irradiation of post-photomorphogenic seedlings is conducted in an open-air growth system. In this system it is clear that etiolated and light-treated seedlings exhibit very different phototropic responses. Additionally, it is evident that the bending is achieved much more quickly, and perhaps most importantly, nutation during bending can be visualized due to the lack of media interaction and hypocotyl drag encountered in traditional systems. Further exploration of the mechanisms underlying the initiation of bending and nutation following blue light irradiation using this open air system will allow a better understanding of how asymmetrical auxin distributions are generated.
IAA oxidation plays important role in plant morphogenesis
Presenter: Jun Zhang Status: Graduate Student
Authors: Jun Zhang, Jinshan Ella Lin, Candace Prichard, Chinchu Harris, Fernanda Mastrott, Fan Wu, Joshua J. Blakeslee, Wendy Ann Peer
Abstract: Auxin oxidation plays important role in auxin homeostasis in Arabidopsis root. We identified AtDAO1 and AtDAO2, two homologues of rice IAA oxidase OsDAO in Arabidopsis by protein sequence alignment. Both of them are 2-oxoglutarate/iron dependent dioxygenase and share a similarity of more than 70% to OsDAO. Recombinant AtDAO1 (DAO1) and AtDAO2 (DAO2) were expressed in E.coli and purified. LC-MS analyses of enzyme assays show that Arabidopsis DAO1 and DAO2 can oxidize IAA to oxIAA in vitro at pH 7.4. The promoters of both genes were found to contain cis-acting elements that are involved in stress responses. Further, we analyzed the expression level of DAO1 and DAO2 in response to different abiotic stress from microarray data sets (Kilian et al., 2007). The results show that DAO1 and DAO2 both respond to multiple abiotic stressors, including salt, drought and UV-B. These results wasconfirmed by qRT-confirmed by qRT-PCR and both of them show stress related phenotypes. These results indicate that DAO1 and DAO2 might have a crucial function in stress acclimation by adjusting auxin homeostasis.
Identifying the role of canonical Wnt/ β-catenin signaling pathway during osteogenic differentiation and the skeletal development in Hutchinson-Gilford Progeria Syndrome
Presenter: Ji Young (Julie) Choi Status: Graduate Student
Authors: Ji Young Choi, Joseph P. Stains, Kan Cao
Abstract: The human bone homeostasis is maintained through constant bone remodeling that balances bone formation and bone resoprtion by keeping sufficient levels of osteoblasts and osteoclasts, respectively. The patients with Hutchinson-Gilford Progeria Syndrome (HGPS) have severe skeletal deformities caused by osteolysis and abnormal ossification, which result in high risk of critical bone fractures. HGPS is a premature aging genetic disease that is mostly caused by a point mutation in LMNA gene, which activates a cryptic splice donor site in exon 11 and results in LMNA mutant protein called, Progerin. In this study, we demonstrated that a canonical Wnt/ β-catenin signaling pathway, one of the major signaling cascade involved in bone cell development, has a role in defective osteogenic differentiation of human Mesenchymal stem cells (MSC) with HGPS mutation. In-vitro study revealed a suppressed level of active (non-phosphorylated) β-catenin in osteogenic differentiated-iPS-derived HGPS MSC. The inhibitory function of progerin on β-catenin signaling pathway during the osteogensis of HGPS MSC was examined. The overexpression of non-farnesylable progerin in wild-type MSC resulted in higher level of β-catenin than regular progerin (farnesylated)-overexpressed MSC. This finding suggests that the non-farnesylation of progerin potentially restores the low basal β-catenin level in HGPS MSC during osteogenic differentiation, and proposes a possible way to rescue the abnormal bone phenotype of HGPS.
Molecular Basis of Glyphosate Resistance in Giant ragweed (Ambrosia trifida L.)
Presenter: KABELO SEGOBYE Status: Graduate Student
Authors: Kabelo Segobye*, Karthik Ramaswamy Padmanabhan, Burkhard Schulz, Michael Gribskov, Stephen C. Weller
Abstract: Molecular Basis of Glyphosate Resistance in Giant Ragweed (Ambrosia trifida L.). Kabelo Segobye*1, Karthik Ramaswamy Padmanabhan2, Burkhard Schulz1, Michael Gribskov2, Stephen C. Weller2. 1University of Maryland, College Park, MD 20740, 2Purdue University, West Lafayette, IN 47907Giant ragweed (Ambrosia trifida L.) is a competitive annual weed found in agriculture fields and disturbed areas of the Midwestern U.S. The introduction of glyphosate resistant agronomic crops (“Roundup®-ready”) in 1996 provided a new tool to manage giant ragweed. Overreliance and repeated use of glyphosate has resulted in tremendous selection pressure for evolution of glyphosate resistant weeds and specifically giant ragweed in Indiana. This study was conducted to investigate the mechanism(s) of resistance to glyphosate and determine genes responsible for glyphosate resistance in glyphosate resistant plants. We hypothesize that the basis of resistance in the Indiana giant ragweed biotype is related to reduced translocation of glyphosate and this resistance leads to a fitness loss in the resistant biotype. Our research involved a direct comparison between glyphosate sensitive (GS) and glyphosate resistant giant ragweed (GR). The two biotypes were compared for response to glyphosate and total mRNA was extracted from leaf disks of untreated and glyphosate treated leaves over a time-course of 0 to 6 hours after herbicide application. The transcriptome of sensitive and resistant giant ragweed biotypes were compared. Results showed that GR plants had a unique response when treated with glyphosate, exhibiting initial rapid necrosis of mature leaves within 12 hours of treatment. GR plants do not die from a glyphosate treatment but resume normal-growth from axillary meristems and reproduce. The progression of the response and symptoms resemble a typical hypersensitive response similar to that observed on some plants after pathogen attack. GS plants do not exhibit rapid leaf necrosis but their leaves become chlorotic, then necrotic and plants die over a 2-3 week period. We have identified a list of genes that were differentially expressed between the two biotypes as the first step in identifying genes responsible for the glyphosate resistance observed. GR plants will persist in the current cropping systems if glyphosate continues to be the main weed control tool.
Programmed -1 Ribosomal Frameshifting in the Encephalitis Family of Alphaviruses
Presenter: Joseph Kendra Status: Graduate Student
Authors: Joseph A. Kendra, Cynthia de la Fuente, Kylene Kehn-Hall, Jonathan L. Jacobs, and Jonathan D. Dinman
Abstract: The new world encephalitic alphaviruses – Eastern, Western and Venezuelan Equine Encephalitis viruses (EEEV, WEEV, and VEEV) are implicated in outbreaks of acute encephalomyelitis. Little is known about the virulence and replication mechanisms of these viruses, presenting significant obstacles to the development of effective therapeutics. A programmed -1 ribosomal frameshift (-1 PRF) signal has been described in the alphavirus structural gene, 6K, whose frameshift product is thought to play a role in viral egress. Here, we have characterized the -1 PRF signals of these three viruses, and probed the importance of frameshifting with regard to viral replication and virulence. The ability of computationally predicted -1 PRF sequences from EEEV, VEEV and WEEV to promote efficient frameshifting was assayed in HeLa and U8-MG astrocyte cell lines using standard dual reporter vectors. Frameshifting was ablated by mutation of the canonical “slippery sites” present in these sequences. Additional controls to probe for cryptic promoters, splice site, or IRES elements in these sequences were all negative, consistent with the hypothesis that these are true -1 PRF signals. While a recombinant live VEEV strain TC8C virus harboring slippery site mutations resulted in only a small delay in viral replication kinetics in both BHK and C3/36 mosquito cells, the frameshift mutant almost completely abrogated TC83 lethality in mouse infection models. These results suggests that, while frameshifting does not exert a significant influence on alphavirus replication, it has a strong effect on virulence. This latter observation suggests the potential utility of -1 PRF abrogation for the production of live attenuated vaccine strains. To better understand these targets, the -1 PRF stimulating RNA structures of EEEV, VEEV and WEEV have been resolved using chemical probing and primer extension methods. Ongoing studies are employing a mutagenesis-based approach to destabilize these key elements of the -1 PRF signals, the results of which could potentially lay the foundation for the development of live attenuated vaccines.
Structure and Function of Plant ABCB Transporters
Presenter: Mark Jenness Status: Graduate Student
Authors: Mark Jenness and Angus Murphy
Abstract: ATP-binding cassette (ABC) transporters are a ubiquitous superfamily of proteins that use ATP hydrolysis to drive the transport of a variety of substrates across membranes. Structural modeling reveals that plasma membrane localized plant and mammalian ABC subfamily B (ABCB) transporters share a high degree of structural conservation. However, plant ABCB transporters function in the relatively specific transport of aliphatic and aromatic organic acids while mammalian homologues are usually associated with the transport of a wide variety of hydrophobic drugs. A unique member of the ABC transporter family, the cystic fibrosis transmembrane conductance regulator (CFTR)/ABCC7, is an ATP-gated chloride ion channel that regulates ion homeostasis in mammalian epithelial cells. Recently, the Arabidopsis auxin transporter ABCB19 was shown to have ion channel activity which was inhibited by the chloride channel blocker NPPB. Additionally, ABCB transporter mutants show decreased root growth when grown on media containing sodium chloride. CFTR requires a physical interaction with the FK506-binding protein 38 (FKBP38) for folding at the endoplasmic reticulum membrane and subsequent trafficking to and stability on the plasma membrane. Like CFTR, a subset of plant ABCB transporters also require an FK506-binding protein (FKBP42) for proper folding, trafficking and activity. These parallels suggest ABC transporter regulation is highly conserved and that substrate transport and channel activity may be part of the same mechanism. To test these concepts, Arabidopsis ABCB transporters and CFTR are being expressed heterologously in Lactococcus lactis and in planta and analyzed for substrate specificity, protein-protein interactions, and functionality.
X-DC as a model ribosomopathy: how defects in ribosome structure affect translational fidelity and gene expression
Presenter: Carol Vieira Status: Graduate Student
Authors: Carolina Marques dos Santos Vieira, Ashton T. Belew, Najib El-Sayed and Jonathan D. Dinman
Abstract: X-linked Dyskeratosis Congenita (X-DC) is a fatal ribosomopathy with a pleiotropic presentation, including anemia, nail dystrophy, connective tissue defects, shortened telomeres and progeria. X-DC is caused by a mutation in DKC1, the gene encoding dyskerin, an RNA binding protein that guides rRNA pseudouridylation. This function is conserved in eukaryotes, from yeast (where the gene is called CBF5) to humans. In published work, we have shown that rRNA pseudouridylation deficient ribosomes have decreased affinity for RNA ligands. Functionally, this defect promotes increased rates of programmed -1 ribosomal frameshifting (-1 PRF), and increased fidelity of stop codon recognition (Jack et al., 2011). These two phenomena lead to increased rates of mRNA degradation through nonsense-mediated mRNA decay (NMD, Belew et al., 2014). We hypothesize that mRNAs that are substrates for NMD, due to the presence of -1 PRF signals or upstream open reading frames, will have a globally lower abundance in the mutant strain. In the current work, RNA-seq experiments were performed to evaluate the global effects of rRNA hypopseudouridylation on gene expression using the yeast-based model. At this juncture, we are analyzing the data to identify differentially expressed genes (DEGs) and the molecular pathways. The list of DEGs will be compared with a list of mRNAs that are predicted to harbor -1 PRF signals, upstream open reading frames, and other properties that may render them substrates for NMD.
Methylene blue alleviates nuclear and mitochondrial abnormalities in Hutchinson-Gilford progeria syndrome
Presenter: Zhengmei Xiong Status: Staff
Authors: Zheng-Mei Xiong, Ji Young Choi, Kun Wang, Haoyue Zhang, Zeshan Tariq, Di Wu and Kan Cao
Abstract: Hutchinson-Gilford progeria syndrome (HGPS), a fatal premature aging disease, is caused by a single nucleotide mutation in the lamin A gene. Previous reports have focused on nuclear phenotypes in HGPS cells, and the potential contribution of the mitochondria, a key player in normal aging, remains unclear. Using high-resolution microscopy analysis, we demonstrated a significantly increased fraction of swollen and fragmented mitochondria and a marked reduction in mitochondrial mobility in HGPS fibroblast cells. Notably, the expression of PGC-1a, a central regulator of mitochondrial biogenesis, was inhibited by progerin. To rescue mitochondrial defects, we treated HGPS cells with a mitochondrial-targeting antioxidant methylene blue (MB). Our analysis indicated that MB treatment not only alleviated the mitochondrial defects but also specifically rescued the hallmark nuclear abnormalities in HGPS cells. Additional analysis suggested that MB treatment released progerin from the nuclear membrane, rescued perinuclear heterochro-matin loss and corrected misregulated gene expression in HGPS cells. Together, these results demonstrate a role of mitochondrial dysfunction in developing the premature aging phenotypes in HGPS cells and suggest MB as a promising therapeutic for HGPS.
A novel isoform of lamin A/C
Presenter: Emily DeBoy Status: Undergraduate Student
Authors: E. DeBoy, M. Puttaraju, Manjula Kasoji, Parthav Jailwala, T. Misteli
Abstract: Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disorder caused by a point mutation in the LMNA gene encoding lamins A and C, two structural proteins of the nuclear envelope. The mutation (C1824T) activates a cryptic splice site in exon 11, causing a 150 bp deletion in the LMNA mRNA. As a consequence, a lamin A isoform, progerin, is generated which is permanently farnesylated due to the removal of an endoproteolytic cleavage site located within the deleted region. During RNA-seq analysis, we discovered a novel LMNA isoform, LMNAd447. The d447 isoform of LMNA uses the same alternative 5’ splice site located in exon 11 which is activated in classical HGPS patients, however, it utilizes a 3’ splice site in the 3’ untranslated region, leading to loss of exon 12 and a 447 bp deletion. LMNAd447 is detected in both normal and HGPS patient cells at very low levels of ~1-3% of mutant LMNA transcripts in HGPS patient cells. Using lentiviral expression we show that LMNAd447 is translated, properly processed and localizes to the nuclear envelope. Preliminary results indicate that overexpression of LMNAd447 in wild type fibroblasts is not sufficient to trigger the HGPS cellular phenotypes typical for patient cells, including nuclear blebbing. The function of LMNAd447 remains unknown.
Coagulation profile dynamics in pediatric patients with Cushing’s syndrome – a prospective observational comparative study
Presenter: Leah Status: Undergraduate Student
Authors: Leah Birdwell, Maya Lodish, Amit Tirosh, Prashant Chittiboina, Meg Keil, Charlampos Lyssikatos, Elena Belyavskaya, Richard Feelders, Constantine A. Stratakis
Abstract: Cushing's syndrome (CS) has been associated with hypercoagulability and thromboembolic events in adults, however, data in pediatric patients is lacking. At the NIH Clinical Center we have experienced clinically significant thromboembolic events in patients with active CS, including an ischemic stroke in one and 2 patients who developed thrombi in a central venous catheter. To better understand this pathophysiological mechanism, we evaluated several key coagulation elements in children with CS before and after intervention and in a control group consisting of normocortisolemic patients. This was a prospective observational study, including 54 consecutive pediatric patients, mean age of 15.1± 3.9 years, that were admitted to the National Institutes of Health for surgical intervention of confirmed CS. Coagulation profiles were taken before intervention and 6-12 months following surgery (either transsphenoidal surgery (n=51), adrenalectomy (n=1), or removal of ectopic ACTH secreting pulmonary carcinoid (n=1). A control group (n=18) of normocortisolemic children (mean age 13.7±3.6 years) was used for comparison. Data were described using simple descriptive statistics and are presented as mean ± standard deviation (SD). Data were compared using t-tests, or Wilcoxon rank-sum test, as appropriate, using SPSS 20. A two-sided P≤0.05 was considered statistically significant. At baseline, patients with CS had higher levels of the endogenous procoagulant FVIII when compared to controls, (145 IU/dL ±84 vs. 99 ±47, p=0.04). In addition, 6-12 months following surgical cure of CS, FVIII levels decreased significantly to 111 ±47, p=0.05. At baseline, patients with CS had higher levels of the antifibrinolytic α2-antiplasmin (A2AAT), 96% ±17 vs. 82 ±26, p= 0.015, and as expected, after surgical intervention the A2AAT levels decreased to 82 ± 24, p <0.001. Endogenous anticoagulants were all higher in CS vs control at baseline, including proteins C (138% ± 41 vs 84±25, p<0.001), protein S (94%±19 vs 74±19, p=0.001, and antithrombin III (AT-III) (96% ±18 vs 77±13, p<0.001). Following surgical intervention, levels of proteins C and S as well as AT-III significantly decreased (P<0.001). 24 hour urinary free cortisol levels positively correlated with FVIII levels, r=0.43, p=0.004. In conclusion, our data shows that patients with CS had elevated procoagulants, antifibrinolytics, and anticoagulants at baseline compared to normocortosolemic control children, and significant decreases in FVII, A2AAT, protein C, protein S, and AT-III were all seen in patients following surgical cure of CS. The degree of hypercortisolism correlated with the levels of procoagulant. This data confirms the hypercoaguable state in children with endogenous hypercortisolemia, and has potential implications for taking additional clinical precautions for the prevention of thrombi in these vulnerable patients.
Expression Optimization and Efficacy Analysis of Cell-Penetrating Peptide-Green Fluorescent Protein Fusions
Presenter: Mackenzie Walls Status: Undergraduate Student
Authors: Mackenzie T. Walls, Zifan Gong, Amy J. Karlsson
Abstract: Cell penetrating peptides (CPPs) are small peptides that have the unique ability to transport through cell membranes through passive or active transport mechanisms. CPPs have the potential to deliver antifungal cargo, including proteins, specifically to fungal pathogens, but the expression of CPP fusion proteins can be challenging. Our work focuses on improving the expression of CPP-green fluorescent protein (GFP) fusion proteins in Escherichia coli cells by analyzing the effects of three variables: temperature, incubation time, and concentration of an inducer. We evaluated the expression of two CPP fusions (pVEC and NPFSD) to GFP with and without a peptide linker. A temperature of 37°C for 8 hours with an inducer concentration of 0.05 μM tended to produce the best expression levels, though the ideal conditions varied for each construct tested. Furthermore, the inclusion of the peptide linker in the fusion protein significantly enhanced expression without affecting its efficacy in Candida albicans. Standards for the optimization of CPP-protein fusions will provide a basis for efficient large scale production of these biomolecules, thereby enhancing the viability of their use as therapeutics.
FRTL-5 Rat Thyroid Cells Release Thyroglobulin (Tg) Sequestered in Exosomes: A Possible Novel Mechanism for Tg Processing in the Thyroid
Presenter: Pavel Alexandrovich Vlasov Status: Undergraduate Student
Authors: Pavel A. Vlasov, Sonia Q. Doi, and Donald F. Sellitti
Abstract: Exosomes are 30-100 nm, membrane-bound vesicles containing specific cellular proteins, mRNAs, and microRNAs that take part in intercellular communication between cells. Exosome secretion into ovarian follicles has been observed but whether the cells lining thyroid follicles secrete exosomes, and what those exosomes contain is not yet known. In the present study, FRTL-5 rat thyroid cells were grown to confluence and received medium containing either thyroid stimulating hormone (TSH), exogenous bovine thyroglobulin (Tg), or neither additive for 24 or 48 hours followed by collection of spent medium and ultracentrifugation to isolate small vesicles. Transmission electron microscopy and Western blotting for CD63 and CD9 suggested the presence of exosomes. Western blotting using a monoclonal Tg antibody revealed a Tg-positive band at ~330kDa (the expected size of monomeric Tg) with a higher density in TSH-treated than in untreated cells. These results are the first to show that normal thyroid cells produce exosomes and confirm the exosomal sequestration of un-degraded Tg. Given the recent findings of regulatory roles for Tg, these results suggest that Tg sequestration into exosomes may be a key requirement for the gene-regulatory actions of Tg.
Generation of a novel fibroblast growth factor dependent binary expression system using CRISPR/Cas9 based genome editing in Drosophila
Presenter: Amy Zhou Status: Undergraduate Student
Authors: Amy Zhou, Lijuan Du, Dr. Sougata Roy
Abstract: Fibroblast Growth Factor (FGF) is a conserved signaling protein essential for metazoan development. In Drosophila FGF acts as a positional cue that induces the tracheal cells to migrate toward the FGF sources and thereby patterns the tracheal tissue into an extensive branched network. This branched tubules transport oxygen to cells. To precisely understand how cells communicate with the FGF signals and how FGF signaling organizes tracheal cells into a branched tubular network, it is necessary to simultaneously manipulate gene activities in both the FGF source and the recipient tracheal cells. This is a highly challenging task because the gene expression systems that can specifically mark only the cells in the FGF source are unavailable and the regulatory sequences that control the dynamic expression of fgf are not known. To achieve this goal, present research aims to generate a new LexA/lexO driven binary transcription system in Drosophila where lexA is expressed under the spatio-temporal control of the regulatory elements of the fgf gene, branchless (bnl). The LexA/lexO is a binary transcriptional system applied from bacteria. When fused with a transcriptional activation domain from eukaryotic species, LexA transactivator activates expression of genes downstream of LexA operator (lexO) sites. This study aims to use the CRISPR/Cas9 based genome editing technique to replace the first exon of the bnl gene with the coding sequence of lexA transactivator. This can be achieved by introducing two guide RNAs targeting either end of the first exon of bnl, as well as a replacement donor with lexA for homology directed repair. We expect that the genome edited fly will express lexA in the same spatio-temporal pattern of the bnl gene and will be extremely useful to manipulate transgene expression in the fgf signal source. Most importantly, this system will enable us to simultaneously manipulate gene expression in two different tissues (trachea and fgf source) by combining the lexA/lexO system with the other available binary systems such as Gal4/UAS. This will broaden the experimental alternatives and be extremely beneficial for future research.
Genetic screening of Uveal Coloboma and other developmental ocular defects in Zebrafish via CRISPR-Cas9 mutagenesis
Presenter: Iain S. Forrest Status: Undergraduate Student
Authors: Iain Forrest, Dr. Sunit Dutta, Dr. Brian Brooks
Abstract: Interruptions in the closure of the optic fissure can cause structural abnormalities of the eye, called colobomas. In humans, colobomas occur in 1 in 2077 live births, with more than 50% having another ocular disorder other than coloboma, including strabismus or amblyopia. Closure of the fissure depends on proximity of the ends of the immature structure and specific signaling. Previously, this lab performed a microarray analysis to identify 250 genes that vary in expression over developmental time of a mouse eye as the optic fissure closes and fuses. Here, as the beginning of a long-term genetic screening of many candidate genes, we investigated atoh7 (math 5) and phb1 genes using CRISPR/ Cas9 and morpholinos for targeted mutagenesis. Prohibitin morphant fish exhibited coloboma and microphthalmia, consistent with this lab’s previous findings. Atoh7 knockout fish showed ocular malformations such as anophthalmia, microphthalmia and ectopic ear-eye reversal. Further investigation is needed to determine knockout specificity via sequencing. Phenotypic analysis of F1 will be conducted in this month.
Investigating the Role of Translational Recoding in Stress Response
Presenter: Avan Antia Status: Undergraduate Student
Authors: Avan Antia, Vivek M. Advani, Jonathan D. Dinman
Abstract: Translational gene regulation involves innumerable processes that monitor the quality and quantity of the mRNA. Programmed -1 Ribosomal Frameshift (-1 PRF) is one such process. First described in viruses, -1 PRF signals have been identified and characterized in higher eukaryotes as cis-acting elements that modulate gene expression. Unlike viral frameshift signals, a ‘genomic’ -1 PRF signal stochastically directs a translating ribosome to a pre-mature termination codon (PTC), thus functioning as an mRNA destabilizing element via the nonsense mediated mRNA decay (NMD) pathway. Computational analysis predicts that approximately 10% of human genes contain at least one significant -1 PRF signal. Using a bicistronic reporter system, we have identified functional -1 PRF signals encoded by five human genes: Poly [ADP-ribose] polymerase 1 (PARP1), RAS p21 protein activator 2 (RASA2), RAS p21 protein activator 4 (RASA4), Autophagy-related gene 7 (ATG7), and Eukaryotic Translation Initiation Factor 2B, Subunit 3 (EIF2B3). These genes are involved in cellular stress response. Interestingly, knockdown of miRNA processing pathway alters the rates of -1 PRF, suggesting sequence specific regulation of -1 PRF promoted by these sequences. The involvement of these genes in cellular stress responses suggests that dysregulation of -1 PRF will affect global gene expression, leading us to hypothesize that -1 PRF plays a role in stress response.
Regulation of Cell Signaling by Programmed -1 Ribosomal Frameshifiting and miRNA
Presenter: Tania Mamdouhi Status: Undergraduate Student
Authors: Tania Mamdouhi, Liya Ketema, Elizabeth Reis
Abstract: Programmed -1 Ribosomal Frameshifting (-1 PRF) is a mechanism of post-transcriptional gene regulation. It was first discovered in Rous sarcoma virus (RSV)1 and later in many RNA viruses as a mechanism to maintain a proper ratio of structural to enzymatic proteins important for viral particle assembly2. Computational analysis suggests that approximately 10% of human genes are regulated by -1 PRF. Recent works have explored the role of -1 PRF signals as mRNA destabilizing elements through nonsense mediated mRNA decay (NMD) pathway in eukaryotic cells3. As a part of the First Year Innovation and Research Experience (FIRE) - Found in Translation (FIT) research stream we have identified functional -1 PRF signals in mRNAs encoding human Eukaryotic Initiation Factor 1A-X (eIF1AX), Mitogen Activated Protein 4 Kinase Kinase Kinase (MAP4K3) and Mitogen Activated Protein 4 Kinase Kinase Kinase Kinase Kinase (MAP4K5). While eIF1AX is a part of translational initiation complex binding, MAP4K3 and MAP4K5 mRNAs encode for key effectors in cell signaling. Preliminary data also shows that -1 PRF signals in these genes affect knockdown of the cellular pseudouridine synthase-dyskerin, a condition that is similar to the disease X-linked Dyskerotosis Congenita (X-DC)4. Additionally, knockdown of miRNA processing pathway also alters the -1 PRF rates promoted by these sequences. This suggests that dysregulation of -1 PRF and/or miRNA transcription affects the expression of these proteins that may contribute to disease pathogenesis. References: 1. Jacks, T., Madhani, H. D., Masiarz, F. R. & Varmus, H. E. Signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region. Cell 55, 447–458 (1988). 2. Dinman, J. D. Mechanisms and implications of programmed translational frameshifting. Wiley Interdiscip. Rev. RNA 3, 661–733. Belew, A. T., Advani, V. M. & Dinman, J. D. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast. Nucleic Acids Res. 39, 2799–808 (2011). 4. Jack, K. et al. rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells. Mol. Cell 44, 660–6 (2011).
Regulation of Eukaryotic Translational Initiation by Programmed -1 Ribosomal Frameshifting.
Presenter: Arvin Massoudi Status: Undergraduate Student
Authors: Arvin Massoudi, Isabella Swafford, Jessica Stimely, Theodore Nikolaitchik, Adam Kellerman, Savannah Speir, Vivek Advani, and Jonathan D. Dinman
Abstract: Translational Recoding refers to exception to the rules of translation. Programmed -1 ribosomal frameshifting (-1 PRF) is one such translational recoding mechanism first identified in some RNA viruses and later described in higher eukaryotes(1). A canonical ‘genomic’ -1 PRF signal directs an elongating ribosome to a -1 frame pre-mature termination codon (PTC), there by resulting in rapid degradation of the message through nonsense mediated mRNA decay (NMD) pathway. There exist an inverse relationship between -1 PRF efficiency and mRNA abundance(2). In silico analysis predicts that approximately 10% of genes in humans have at least one predicted -1 PRF signal(3). In this work we have identified functional frameshift signals in mRNAs of human translational initiation factors eIF3E, eIF4B, eIF4G, eIF5B and eIF2AK4. These proteins are associated with some cancers and play a significant role in development and aging(4). Interestingly, knockdown of the cellular pseudouridine synthase-dyskerin, a condition that is similar to the disease X-linked Dyskeratosis Congenita (X-DC), alters the -1 PRF rates promoted by these sequences. This suggests that dysregulation of -1 PRF affects the expression of these proteins that leads to disease pathogenesis. Additionally, computational analysis also predicts certain miRNAs that may interact and alter the rates -1 PRF promoted by these sequences. The proposed work will increase our understanding of -1 PRF and gene regulation. References: 1. Jacobs JL, et. al. Identification of functional, endogenous programmed -1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae. Nucleic Acids Res [Internet]. 2007 Jan 12 [cited 2015 Mar 17];35(1):165–74. 2. Belew AT, et. al. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast. Nucleic Acids Res [Internet]. 2011 Apr 1 [cited 2014 Oct 14];39(7):2799–808. 3. Belew AT, et. al. PRFdb: a database of computationally predicted eukaryotic programmed -1 ribosomal frameshift signals. BMC Genomics [Internet]. 2008 Jan [cited 2014 Oct 15];9(1):339. 4. Silvera D,et. al. Translational control in cancer. Nat Rev Cancer [Internet]. Nature Publishing Group; 2010 Apr 1 [cited 2015 Aug 26];10(4):254–66.
The JAK-STAT pathway is regulated by miRNA mediated Ribosomal Frameshifting.
Presenter: Yousuf A. Khan Status: Undergraduate Student
Authors: Yousuf A. Khan, Vivek M. Advani, Zachary Flickinger, Jonathan D. Dinman
Abstract: Programmed -1 ribosomal frameshifting (-1 PRF) is a translational recoding mechanismwhereby a translating ribosome is induced to slip one nucleotide in the 5’ directionby a complex mRNA cis-acting element (1). In the context of cellular mRNAs, such events overwhelmingly direct ribosome to premature termination codons (PTC), rendering these mRNAs substrates for the nonsense mediated mRNA decay (NMD) pathway(2). Approximately 10% of all human genes have at least one predicted significant -1 PRF motif, suggesting that -1 PRF is extensively used to regulate gene expression in human cells(3). Here we have characterized the -1 PRF signals in the mRNAs encoding human Janus Kinase 2 (JAK2) and Signal Transducer and Activator 1 (STAT1), both of which are involved in JAK/STAT intracellular signaling. Additionally, preliminary data suggests that JAK2-mediated -1 PRF is inhibited by two miRNAs in a sequence specific manner. We seek to further characterize the role of -1 PRF and the miRNAs in regulating the JAK-STAT pathway. This study will serve to understand the role of translational recoding in immune signaling.
Translational Recoding in Cancer
Presenter: Jacob Greenspan Status: Undergraduate Student
Authors: Jacob Greenspan, Alyssa Gabay, Rohan Singh, Ajay Chatim, Kali Murphy and Vivek Advani
Abstract: Translational Recoding refers to exception to the rules of canonical translation. Programmed -1 Ribosomal Frameshifting (-1 PRF) fits this paradigm. -1 PRF was first discovered in viruses1 and later described in other eukaryotic genes. While viruses use -1 PRF for proteomic expansion, recent work suggests that this mechanism is used to regulate cellular gene expression in eukaryotes23. There are predicted -1 PRF signals in mRNAs encoding tyrosine-protein kinase ABL1 isoform A (ABL1), Homo Sapiens cadherin 16 (CDH16), BCL2-like 11 (BCL2L11), and BCL CLL/lymphoma 9 (BCL9) genes. The BCL2L11 gene is known to encode for a proteins that act as apoptotic activators. The BCL9 gene is associated with B-cell acute lymphoblastic leukemia—a type of cancer that affects the white blood cells of the bone marrow. CDH16 plays a key role in maintaining the cohesion of the kidney tissue. It is also linked to cancer, as lack of expression is associated with renal cell carcinoma that stems from epithelial kidney tubular cells. ABL1 is associated with responding to DNA damage caused by leukemia-related stress. The hypothesis is that these genes have functional -1 PRF signals and contribute to regulation of expression in these genes. Through bicistronic reporter system functional -1 PRF signals were identified in these genes. One particular gene site on the BCL9 gene has been shown to exhibit -1 PRF at a rate of approximately 2 percent. If these predicted sequences are conclusively proven to be functional, then further understanding of gene regulation in cancer may be obtainable. References: 1. Jacks, T., Madhani, H. D., Masiarz, F. R. & Varmus, H. E. Signals for ribosomal frameshifting in the rous sarcoma virus gag-pol region. Cell 55, 447–458 (1988). 2. Belew, A. T., Advani, V. M. & Dinman, J. D. Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast. Nucleic Acids Res. 39, 2799–808 (2011). 3. Plant, E. P., Wang, P., Jacobs, J. L. & Dinman, J. D. A programmed -1 ribosomal frameshift signal can function as a cis-acting mRNA destabilizing element. Nucleic Acids Res. 32, 784–90 (2004).
Translational Recoding in Embryonic and Cardiovascular development
Presenter: Mina Griffioen Status: Undergraduate Student
Authors: Mina Griffioen, Amanda Lee, Samantha McGeehon, Sanad Naber, Michael Wong
Abstract: Translational recoding, first described in viruses, has recently emerged as an important mechanism of post-transcriptional gene regulation. Prior studies from our laboratory have explained how Programmed -1 Ribosomal Frameshifting (-1 PRF) fits within this paradigm. Analysis of predicted -1 PRF signals across 20 genomes suggests that it is a universal mechanism1. The overwhelming majority of “genomic” -1 PRF events are predicted to direct translating ribosomes to premature termination codons. We have demonstrated that these can function as mRNA destabilizing elements through the Nonsense-Mediated mRNA Decay (NMD) pathway2. As a part of the First Year Innovation and Research Experience (FIRE) - Found in Translation (FIT) research stream, we were able to identify functional -1 PRF signals in human mRNA encoding GATA binding protein 5, Forkhead box L1, Forkhead box J2, Forkhead box I3, and BCL2-Associated Athanogene 3. These genes play a critical role in embryonic organ development and cardiovascular tissue formation (OMIM, n.d.). The frameshift efficiency of each gene is determined by performing a Dual Luciferase assay on Human Embryonic Kidney (HEK) and HeLa cells transfected with the gene. A -1 PRF event on these genes directs ribosomes to a premature termination codon (PTC), resulting in truncated proteins which become substrates to the NMD pathway. Previous work has shown that dysregulation of -1 PRF rates results in altered gene expression that may contribute to human disease. The genes in consideration have been associated with human diseases such as atrioventricular septal defect (AVSD), breast cancer, alzheimer's, or cardiomyopathy. Hence -1 PRF promoted by these sequences can be potential target for gene therapy. In silico alignments suggests potential miRNAs that may interact with the -1 PRF sequences to regulate their expression via -1 PRF.