Day 1 :
Keynote Forum
W. Richard McCombie
Cold Spring Harbor Laboratory, USA
Keynote: The impact of very long sequencing reads on our view of genomes and transcriptomes
Time : 09:55-10:20
Biography:
W. Richard McCombie, Ph.D., is a Professor at Cold Spring Harbor Laboratory and the Watson School of Biological Sciences. He is the Director of the Stanley Institute of Cognitive Genomics at Cold Spring Harbor Laboratory in New York and Program Leader for the Cancer Genetics at CSHL. He received his B.A. in Biology from Wabash College and his Ph.D. in Cellular and Molecular Biology in the Health Sciences from the University of Michigan. As a Senior Staff Fellow in Craig Venter’s section at the National Institutes of Health, he was a leader of one of the first groups to carry out large-scale automated sequencing of genomic DNA and helped to organize the first large-scale EST (expressed sequence tag) sequencing projects. He has been on the faculty at CSHL since 1992 and was named a professor in 2001. Professor McCombie’s lab has contributed to the efforts to sequence the genomes of several organisms, including the flowering plant Arabidopsis thaliana, the fission yeast S. pombe, rice, mouse and human. The McCombie lab has also focused considerable effort on the development of methods and strategies for genome analysis. These have included development of exome sequencing methyl filtration in plants and as well as contributing to the first single cell analysis of cancer. An author of many published papers in the field of genomics, Professor McCombie has developed and taught courses on applications of genome sequencing and on genomics and proteomics at the Watson School of Biological Sciences at CSHL. He has also organized an intensive DNA sequencing course at CSHL. Professor McCombie and colleagues are currently developing optimized ways to carry out de novo assemblies of plant genomes using next generation sequencing approaches and newer, long read base sequencing instruments. They are also using these approaches to determine the salient variation contributing to human disease.
Abstract:
The development of short read sequencing on Illumina platforms has created an explosion of information about genome structure as well as transcriptome variability. However, the short reads have led to limitations in understanding of both the genomes and transcriptomes. In general terms the short reads require mapping back to a reference genome rather than doing an assembly, de novo, from the raw data. This is inherently problematic since structural rearrangements which are very important in instances like cancer may be lost since they are not represented in the reference. In addition, there are regions of the genome which do not map well and are difficult to interpret their structure based on the short reads. Likewise, while counting reads from transcriptome data has provided powerful statistical analyses of the variability of gene expression, the short reads give a limited view of alternate splice isoforms. Very long reads (in excess of 10kb average) as are now possible on the Pacific Biosciences platform and are shedding new light on the possible impact of these limitations. We have sequenced the genome of a breast cancer cell line SKBR3 as well as carried out analysis of transcriptome data from the same cell line using very long reads. This has shown a very different view of the genome than we see with short reads including a number of structural rearrangements, insertions and deletions that appear to be missed in the short read sequence and a significant number of gene fusions that are missed in the transcriptome analyses. These discrepancies and our further studies of them and how they might impact our understanding of the biology of this cancer cell line will be elaborated.
Keynote Forum
Glen M Borchert
University of South Alabama, USA
Keynote: Fully mining RNA-Seq data for noncoding RNA analyses
Time : 09:30-09:55
Biography:
Glen M Borchert has completed his PhD in Genetics from University of Iowa in the year 2006. He did his first postdoctorate in Structural Biology from University of California in the year 2008 and the second one in Immunology from Illinois State University in the year 2012. After which he joined as Assistant Professor in University of South Alabama and currently working as Assistant Professor, Pharmacology in USA College of Medicine. He has been honored by NIH with a Research award to study hypoxia induced mutation. Also, National Science Foundation (NSF) gave him the CAREER award of $533,000 to research miRNA targeting.
Abstract:
Breast cancer is the leading cause of female cancer mortality. Strikingly, the two most widely utilized breast cancer cell lines, primary MCF-7 and metastatic MDA-MB-231 breast cancer cells, have been used in over half of all the breast cancer studies in the primary literature. Since these cell lines differ in several well established ways in terms of morphology, invasiveness and physiological responses, we recently performed a RNA-seq analysis examining both their total RNA and small RNA populations in order to identify novel gene candidates responsible for their phenotypic differences. Having successfully generated over 150 million transcript sequencing reads from these cells, we now have extensive coverage of the mRNA and small RNA transcriptomes for each of these lines allowing us to identify specific regulations responsible for characteristic differences between them. To our surprise, distinct examinations of this data have generated three major new lines of investigation for our research. While we find little to no change in the expressions of over 2,500 human microRNAs between these cell lines, we identify 25 miRNAs significantly overexpressed in MDA-MB-231 cells as well as 19 miRNAs overexpressed in MCF-7s. Strongly corroborating the importance of these miRNAs in breast cancer, 39 of these 45 miRNAs have been previously reported as being directly involved with breast cancer pathology and/or the modulation of breast cancer cellular response to chemotherapeutic agents. As such, we are now actively engaged in determining cellular functions for the six new miRNAs we find differentially expressed between MCF-7 and MDA-MB-231 cells likely playing uncharacterized roles in breast cancer pathology. Further computational analyses of our RNA-Seq data identified over 250,000 A-to-I edit sites primarily located in mRNA 3’ UTRs. When these locations were screened against the list of currently annotated miRNAs we discovered that these A-to-I editing events caused a subset (~5%) of human miRNAs to have significantly altered mRNA complementarities leading us to propose that modulating the targets of miRNAs via mRNA editing plays a direct role in the pathology of many carcinomas. And , in a more recent analysis of our RNA-Seq data we compared the snoRNA derived RNA (sdRNA) expression profiles of MCF-7 and MBA-MD-231 cell lines. Excitingly, we find 13 snoRNAs significantly overexpressed (≥10 fold) in MBA-MD-231 cells as compared to MCF-7s. To our surprise, we found microRNA-like fragments derived from all 13 snoRNAs were expressed in MBA-MD-231s. Moreover, additional experimentation finds small RNA reads from 10 of 13 small RNA-generating snoRNAs are complexed with Ago following immunoprecipitation suggesting their active involvement in RNAi and potential relevance to breast cancer pathology. In summary, we find RNA-Seq provides a comprehensive, quantitative, and unbiased view of RNA sequences allowing for the ready discovery of novel observations unobtainable with previous technologies and that the data generated by a single RNA-Seq can lead to numerous new lines of investigation.
- Track 2: RNA Editing and RNA Interference: Interplay, Track 3: Next Generation Sequencing (NGS) Technologies, Track 4: Disease Interrogation and Applications of RNA-Seq
Location: Continental Ball Room 5 & 7
Chair
Momiao Xiong
University of Texas Health Science Centre, USA
Co-Chair
Yongming Sang
Kansas State University, USA
Session Introduction
Momiao Xiong
University of Texas Health Science Centre, USA
Title: Integrative image and RNA-Seq data analysis
Time : 10:35-10:55
Biography:
Momiao Xiong, Ph. D, Professor, Division of Biostatistics and Human Genetics Center, School of Public Health, the University of Texas Health Science Center at Houston . He obtained his PhD degree from the Department of Statistics at the University of Georgia in 1993 and finished his postdoctoral training in computational biology from the University of Southern California in 1995. His research is in the area of statistical genetics, bioinformatics, machine learning and image analysis.
Abstract:
Emerging integrative analysis of increasingly larger and complex genomic and anatomical imaging data which has not been well developed, provides invaluable information for the holistic discovery of the genomic structure of disease and has the potential to open a new avenue for discovering novel genetic influence on the imaging variation and disease which cannot be identified if they are analyzed separately. Both imaging and genomics generate a huge amount of data that present critical bottleneck in the interactive analysis. A key issue to the success of imaging and genomic data analysis is how to reduce dimensions of both imaging and genomic data. Most previous investigated methods for imaging information extraction and RNA-seq data reduction do not explore imaging spatial information and often ignore gene expression variation at genomic positional level. To overcome these limitations, we extend one dimensional principle component analysis to two dimensional principle component analyses (2DFPCA) and use 2DFPCA scores to represent image data. Unlike the widely used linear regression for modeling association of microarray gene expressions with phenotypes which quantifies the expression level of a gene/transcript by a single number, we develop a multiple functional linear model (MFLM) in which functional principal scores of images are taken as multiple quantitative traits and RNA-seq profile across a gene is taken as a function predictor for assessing the association of gene expression with imaging signals which can take gene splicing and expression variation at genomic positional level into account. The developed method has been applied to image and RNA-seq data of ovarian cancer and KIRC studies. We Identified 24 and 84 genes whose expressions were associated with imaging variations in ovarian cancer and KIRC studies, respectively. Our results showed that many significantly associated genes with images were not differentially expressed, revealed their morphological and metabolic functions. The results also demonstrated that the peaks of the estimated regression coefficient function in the MFLM often allowed to discover splicing sites and multiple isoform of gene expressions.
Atsushi Shimizu
Iwate Medical University, Japan
Title: Reduction of systematic bias in transcriptome data from human peripheral blood mononuclear cells for transportation and biobanking
Time : 10:55-11:15
Biography:
Atsushi Shimizu graduated from Aoyama Gakuin University, Tokyo, Japan and obtained PhD degree in science from Aoyama Gakuin University in 1999. He then worked as a post-doc with Professor Nobuyoshi Shimizu at Keio University School of Medicine, Tokyo, Japan. He was an Associate Professor at the Keio University School of Medicine. He is now a Principal Investigator at Iwate Medical University. Over the last two years, he has developed a research project with his group focusing on the large-scale genome cohort study aiming to characterize the genome, epigenome and transcriptome analysis.
Abstract:
Transportation of samples is essential for large-scale biobank projects. However, RNA degradation during pre-analytical operations prior to transportation can cause systematic bias in transcriptome data, which may prevent subsequent biomarker identification. In this study, we examined the effectiveness of RNA-stabilizing reagents to prevent RNA degradation during pre-analytical operations with an emphasis on RNA from PBMCs to establish a protocol for reducing systematic bias. To this end, we obtained PBMCs from 11 healthy volunteers and analyzed the purity, yield, and integrity of extracted RNA after performing pre-analytical operations for freezing PBMCs at -80°C. We selected 7 samples from 11 healthy volunteers, and systematic bias in expression levels was examined by RNA-Seq experiments and data analysis. Our data demonstrated that omission of stabilizing reagents significantly lowered RNA integrity, suggesting substantial degradation of RNA molecules due to pre-analytical freezing. RNA-Seq for 25,223 transcripts suggested that about 40% of transcripts were systematically biased. These results indicated that appropriate reduction in systematic bias is essential in protocols for collection of RNA from PBMCs for large-scale biobank projects. Among the seven commercially available stabilizing reagents examined in this study, RNA-Seq experiments consistently suggested that RNALock, RNA/DNA Stabilization Reagent for Blood and Bone Marrow, and 1-Thioglycerol/Homogenization solution could reduce systematic bias. On the basis of the results of this study, we established a protocol to reduce systematic bias in the expression levels of RNA transcripts isolated from PBMCs. We believe that these data provide a novel methodology for collection of high-quality RNA from PBMCs for biobank researchers
Yongming Sang
Kansas State University, USA
Title: Genome-wide transcriptomes integrate animal intra- and inter-organism processes and evoke the ecoimmunological concept in animal health
Time : 11:15-11:35
Biography:
Yongming Sang has completed his DSc and PhD at Nanjing Agricultural University and Kansas State University, respectively. He is a principal investigator and an Assistant Professor currently at Kansas State University. He has published 40 papers in reputed journals and has been serving as a reviewer of multiple journals and grant panel of repute. Main research efforts in his lab focus on immunometabolic regulation underlying animal viral and obesity diseases
Abstract:
Whereas classic immunology deals with animal health primarily in the immune system of a laboratory-based animal species, the emerging discipline of ecoimmunology seeks to understand bona fide immune reactions integrating into intra-organismic systems and interacting with ecological and even evolutionary processes. In this context, non-bias transcriptomic analyses, such as RNA-Seq, provide genome-wide gene expression profiles underlying versatile interaction of the immune system with other systems at intra- and inter-organismic levels. For example, our transcriptomic analysis of porcine macrophages at different activation statuses in response to a viral infection, not only revealed differential expression of multiple immune gene families but also highlighted the involvement of lipid metabolism and even circadian rhythm genes linking to neuroimmune regulation. In addition, our RNA-Seq data in ileal samples of obese rats induced using high-fat diet plus an adenoviral infection showed that not only genes in lipid metabolic pathways but genes in endocrine and immune systems were significantly differentially regulated. Moreover, gene ontology (GO) analysis of the rat ileal RNA-Seq data also revealed that signaling pathways involved in multi-organism interaction were significantly altered in the obese animals, indicating that inter-organism processes (such as gut microbiota alteration) provoke the development of obesity. In sum, as multifaceted interactions centered on immune responses may be intuitive and highlighted in other related studies in animal health, genome-wide transcriptomic analyses provide a non-bias quantifying approach for studying multi-system integration, which may elicit mechanistic studies and therapeutic strategies pertinent to the discipline of ecoimmunology.
Bingsong Zheng
Zhejiang A & F University, China
Title: Identification by deep sequencing and profiling of conserved and novel hickory microRNAs involved in the graft process
Time : 11:35-11:55
Biography:
Bingsong Zheng has completed his PhD in 2003 from Zhejiang University and Postdoctoral studies from INRA. He is the Vice-Dean of School of Forestry & Biotechnology, Zhejiang A & F University. He has published more than 30 papers in reputed journals and has been serving as an Editorial Board Member of repute.
Abstract:
MicroRNAs (miRNAs) play a vital role in plant development and growth through negative regulation of post-transcriptional gene expression. Carya cathayensis (hickory) is an important species for dried nuts and oil in China with high nutritional and economic value. The graft technique is an important strategy for hickory cultivation. To understand the role of miRNAs involved in the hickory graft process, we constructed three small ribonucleic acid (RNA) libraries from hickory rootstock (2 years old) and scion (1 year old) at 0, 7 and 14 days postgrafting. Sequence analysis of the three libraries identified 21 conserved miRNAs belonging to 13 families and 10 novel and 8 potentially novel miRNAs belonging to 15 families. Among these miRNAs, 12 miRNAs were differentially expressed during the graft process in hickory and two-thirds were downregulated. Quantitative real-time polymerase chain reaction (qRT-PCR) validated that 14 miRNAs and their expression trends were similar to the results obtained by Solexa sequencing. Further, a total of 89 target genes for conserved and 26 target genes for novel miRNAs were predicted. This study will help in understanding the roles and regulatory modes of miRNAs involvement in the hickory graft process.
Michael Bonin
IMGM Laboratories GmbH, Germany
Title: RNA Sequencing in degraded tumor RNA samples: Two natural enemies finally reconciled
Time : 11:55-12:15
Biography:
Michael Bonin has completed his PhD in Genetics at the Kassel University. From 2001 to 2014 he served as Head of the Microarray Core Facility located at the Institute of Medical Genetics and Applied Genomics, Medical Faculty, University Tuebingen. Since August 2014 Dr. Bonin is the managing director of IMGM Laboratories, a leading genomic service provider in Europe. He has published more than 120 papers in reputed journals with an overall impact factor of more than 600.
Abstract:
Carved in stone, painted on canvas or scratched in vinyl expressions of human thoughts and feelings have been conserved over generations and been ever since an easily accessible treasure of knowledge and inspiration. When it comes to expression of genes conserved in FFPE or degraded RNA samples, things are different. Given the degradation of RNA due to fixation or other circumstances to shreds and pieces, the scientific treasure of potential insights and discoveries is everything but easy to unearth. Unfortunately, classic approaches often suffer from high sample amount input requirements and highly variable results. These hamper immensely the effective gene expression analysis of challenging FFPE or degraded but nevertheless precious and irreplaceable RNA samples. In the current study strongly degraded total RNA samples from stomach endoscopies of patients with gastric carcinomas should be analyzed for gene expression changes between tumor regions and normal tissue regions. To overcome the described problems, we established a new RNA Sequencing Service workflow. The library preparation with the Illumina TruSeq RNA Access kit served as a key step especially addressing low-quality RNA samples and requiring input amounts of as little as 20-100 ng. RNA was evaluated for usability by the newly established Illumina DV200 quality parameter. The subsequent stranded RNA Access library preparation included a sequence-specific enrichment technology. This enables reproducible transcript capture not compromised by poly-A bias using >4,25000 probes, interrogating >2,10000 targets of 21,415 genes of hg19 and thus covering 98.3% of RefSeq. By this approach, variability was diminished while required sequencing depth was greatly reduced. The IMGM´s FFPE RNA Sequencing Service was expanded by 2×75 bp paired-end sequencing on the NextSeq500 platform and a comprehensive data quality control and analysis on the CLC Bio Genomic Workbench. Sequencing resulted in a high amount of high quality paired end reads and a very high portion could be mapped to the human genome regardless the level of degradation of the underlying RNA samples. The comparison of the resulting data with data from two of the samples which underwent a classical mRNA library preparation protocol showed that the used TruSeq RNA Access protocol enhanced the amount of mappable reads and diminished sequencing of intergenic regions. Thereby paired-end reads could be concentrated on meaningful gene regions in high sequencing depth finally enhancing the focus for detection of significant differential expression levels and fusion genes. Thus, the used approach of the IMGM´s FFPE RNA Sequencing Service powered by the Illumina TruSeq RNA Access kit serves as an efficient step towards gaining so far hidden transcriptomic insights in difficult to access RNA samples.
Glen M Borchert
University of South Alabama, USA
Title: RNA Editing Alters MicroRNA Targeting in Human Breast Cancer
Time : 12:15-12:35
Biography:
Glen M Borchert has completed his PhD in Genetics from University of Iowa in the year 2006. He did his first postdoctorate in Structural Biology from University of California in the year 2008 and the second one in Immunology from Illinois State University in the year 2012. After which he joined as Assistant Professor in University of South Alabama and currently working as Assistant Professor, Pharmacology in USA College of Medicine. He has been honored by NIH with a Research award to study hypoxia induced mutation. Also, National Science Foundation (NSF) gave him the CAREER award of $533,000 to research miRNA targeting
Abstract:
RNA editing by RNA specific adenosine deaminase (ADAR) is increasingly being found to alter microRNA (miR) regulations. Editing of miR transcripts can affect their processing as well as what mRNAs they target. Further, editing of target mRNAs can also affect their complementarily to miRs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes largely unclear. In addition, reports have also identified many miRs to be differentially expressed in cancer though the majority of their targets are also undefined. Here we propose that modulating the targets of miRs via mRNA editing can play a direct role in the pathology of many carcinomas. In order to more accurately characterize the relationship between these two regulatory processes this study examined RNA editing events within mRNAs sequences of two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits modulate miR associations. Computational analyses of RNA-Seq data from the two cell lines identified over 250,000 edit sites within mRNAs, many of which were located in 3’ UTR regions. When these locations were screened against the list of currently annotated miRs we discovered that editing caused a subset (~5%) to have significant alterations to mRNA complementarity. One miR in particular, miR-140-3p has been shown to be abhorrently expressed in many breast cancers. Interestingly, we found that mRNA editing made this specific miR able to target the apoptosis inducing gene DFFA in MCF-7 but not in MDA-MB-231 cells. As these two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and physiological responses, it is feasible that RNA editing could contribute to the phenotypic differences observed between the two cell lines and help explain why an increased incidence of ADAR activity is detected in a number of malignancies. Broadly, these results suggest that the creation of miR targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity.
Sang Ming WANG
University of Nebraska Medical Center
USA
Title: Genome-scale mapping promoter variation through exome sequencing
Time : 12:35-12:55
Biography:
San Ming Wang finished his Master of Medicine during September, 1983—July, 1986 from Shandong Medical University, Jinan, China. He pursued Doctor of Medicine December, during the year December 1989—June, 1994 at Genetic Unit, Swiss Institute for Experimental Cancer Research (ISREC) / University of Lausanne, Switzerland. He worked as Assistant Professor during January, 2004–October, 2009 at Northwestern University. Later he got appointed as Director for Center for Functional Genomics, ENH Research Institute (Now named NorthShore University HealthSystem Research Institute). From October, 2010 – till date, he is working as an Associate Professor at Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center, Nebraska.
Abstract:
Promoter plays essential roles in regulating gene expression. Alteration in promoter sequences can change gene expression, leading to profound impact in evolution, physiology, and disease. Comprehensive analysis of promoter sequences is critical to understand gene expression regulation under biological and pathological conditions. Here we report the development of a method named EPA (Exome-based Promoter Analysis) for the purpose. The method takes the advantage of the random fragmentation used in exome sequencing to obtain the promoter-exon containing sequences. A unique feature of the method is that it preserves the original contents in promoters for sequencing, therefore, allows de novo detection of the changes in promoters without a priori knowledge. This overcomes the weakness in hybridization-based methods as they rely on the normal promoter sequences to design probes. Our evaluation of the method in an exome data set generated from human CD4+ T helper cells shows that the method provides high sensitivity to detect most of the gene promoters, and high specificity to detect the common, rare and novel variants in the detected promoters, TF binding motifs, and TATA boxes. EPA method provides a simple but effective means for genome-scale promoter study.
- Workshop: RNA editing and Next Generation Sequencing
Location: Continental Ball Room 5 & 7
Session Introduction
Glen M Borchert
University of South Albama, USA
Title: RNA editing and Next Generation Sequencing
Time : 13:45-14:30
Biography:
Glen Mark Borchert has completed his PhD in Genetics from University of Iowa in the year 2006. He did his first Postdoctorate in Structural Biology from University of California in the year 2008 and the second one in Immunology from Illinois State University in the year 2012. After which he joined as Assistant Professor in University of South Alabama and currently working as Assistant Professor, Pharmacology in USA College of Medicine. He has been honored by NIH with a Research award to study hypoxia induced mutation. Also, National Science Foundation (NSF) gave him the CAREER award of $533,000 to research miRNA targeting.
Abstract:
RNA editing by RNA specific adenosine deaminase (ADAR) is increasingly being found to alter microRNA (miR) regulations. Editing of miR transcripts can affect their processing as well as what mRNAs they target. Further, editing of target mRNAs can also affect their complementarity to miRs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes largely unclear. In addition, reports have also identified many miRs to be differentially expressed in cancer though the majority of their targets are also undefined. We propose that modulating the targets of miRs via mRNA editing can play a direct role in the pathology of many carcinomas. To address this, we have recently developed new algorithms to identify and analyze RNA edit sites in RNA-Seq data and used these to identify microRNA target sites created and destroyed by deamination of mRNA adenosines. Initial computational analyses of some of our breast cancer RNA-Seq data identified over 250,000 A-to-I edit sites primarily located in mRNA 3’ UTRs. When these locations were screened against the list of currently annotated miRs we discovered that these A-to-I editing events caused a subset (~5%) of human miRs to have significantly altered mRNA complementarities leading us to propose that modulating the targets of miRs via mRNA editing plays a direct role in the pathology of many carcinomas. Broadly, these results suggest that the creation of miR targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity. Furthermore, our results strongly indicate that the creation of miR regulatory sites is a novel (and surprisingly prevalent) function for ADAR activity, and consequently, many miR target sites are only identifiable through the examination of expressed sequences.
- Track 1: Transcriptome analysis and Gene Expression-An Overview, , Track 5: Exploring the complexity of the Transcriptome & Track 6: Refining expression analysis
Location: Continental Ball Room 5 & 7
Chair
Andrey S. Krasilnikov
Penn State University, USA
Co-Chair
Yan Li
The Fourth Military Medical University, China
Session Introduction
Andrey S. Krasilnikov
Penn State University, USA
Title: Transcripts to cleave transcripts: Structure and function of RNA-based Ribonucleases P and MRP
Time : 14:30-14:50
Biography:
Andrey S. Krasilnikov has received his PhD degree from the Institute of Chemical Physics (Moscow, Russia) in 1995. During his postdoctoral training at the UIC and Northwestern University (Chicago, USA) he became interested in enzymes that rely on their RNA moiety for catalysis. He is currently an Associate Professor of Biochemistry and Molecular Biology at the Penn State University. The main focus of his research is the structure and function of the multicomponent eukaryotic catalytic ribonucleoproteins of the RNase P/MRP family. He has authored more than 30 publications in the fields of biochemistry, molecular and structural biology, and physics.
Abstract:
RNA-based enzymes of the Ribonuclease (RNase) P/MRP family are large catalytic ribonucleoprotein complexes. These enzymes are highly unusual as they do not use proteins as their catalytic moiety, but rely on RNA for catalysis. The best known enzyme of the family, RNase P, is found in all three domains of life and is primarily responsible for the maturation of the 5’-end of tRNAs. Bacterial RNase P consists of an RNA component that is capable of cleaving substrates in vitro without any protein participation, and a small protein that is required for activity under physiological conditions. Archaeal and eukaryal RNases P have RNA components that are similar to that in the bacterial enzyme, although the catalytic ability of their RNA is severely diminished. At the same time, the essential auxiliary protein part of the archaeal and eukaryal RNases P grew considerably. The reasons for the increased reliance on proteins in the more evolutionarily advanced RNases P are not well understood. The other, much less studied enzyme of the RNase P/MRP family, is RNase MRP. RNase MRP is universally found in all eukaryotes. It is an essential enzyme that participates in the metabolism of a wide range of transcripts, from rRNA to some mRNAs. Mutations in RNase MRP result in a range of developmental disorders in humans. Structurally RNase MRP resembles eukaryotic RNase P, but it has evolved to have distinct specificity. We will discuss the recent advances in our understanding of the structure, function, and evolution of RNases P and MRP
Marcelo S. Ferro
University Sao Judas Tadeu, Brazil
Title: The mitochondrial transcription factor and the interaction on Cell Aging
Time : 14:50-15:10
Biography:
Marcelo S. Ferro is a nutritionist, former athlete, post graduate in clinical pharmacology, master in aging science (gerontology) in organelles of cardiomyocytes, nutritionist holder of the Paralympic judo team of Brazil, former university professor of biochemistry and nutrition, acts as technical coordinator companies processing of nutritional supplements in Brazil, worked in partnership with the Group of Pain in the Hospital das Clinicas (HC-USP) as Nutritionist for several years in São Paulo, Brazil.
Abstract:
The mitochondria are essential in numerous physiological processes, including energy production, redox potential, modulation of calcium and several metabolic pathways. When the number or mitochondrial activity is insufficient, the human body quickly goes into fatigue due to ATP deficiency. The oxidative capacity of muscle tissue and the preservation of mitochondria depends on the mitochondrial biogenesis that occurs through the transcription factor proliferator-activator receptor-γ coactivator1α (PGC-1α). The oxidative process and the progressive change in the biogenesis of mitochondria have direct influence on the aging of muscle tissue. The regulation of the biogenesis occurs through the PGC-1α combined with nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factors (TFAM). Abnormalities in mitochondria and mutagenesis in mitochondrial DNA (mtDNA) are tied to multi-system degeneration, as well as intolerance to stress, and decreased energy in aging in humans, rats and monkeys. The mitochondrial functions are dramatically altered in heart disease, demonstrating a decrease in expression of PGC-1α, which plays a key role in the coordination of energy metabolism. This process can be reversed by the PGC-1α itself. The identification of compounds capable of activating the transcription of PGC-1α could be part of future therapies to reverse pathologies associated with the decline of this organelle. Morphophysiological and biochemical changes of these organelles directly reflect the physiological performance of all body tissues. Evidence demonstrated that physical activity, both in young and aged is a major ally in mitochondrial biogenesis by activating the transcription of PGC - 1α and that future nutritional interventions may be of great aid in the health and performance of mitochondria. However, further studies are needed in order to understand and clarify this operation, since currently these mechanisms are only partially known.
Yan Li
The Fourth Military Medical University, China
Title: NDRG2, a new estrogen-targeted gene
Time : 15:10-15:30
Biography:
Yan Li has completed her PhD from the Fourth Military Medical University (China) in 2009 and she used to be a Visiting Scholar for Postdoctoral research in Purdue University (USA). Now she is an Associate Professor of the department of Biochemistry and Molecular Biology, the Fourth Military Medical University (China). She has published more than 20 papers in academic journals including J BiolChem, Mol Ther, Breast Cancer Res and Cell Death Dis.
Abstract:
HumanN-myc downstream regulated gene 2(NDRG2) has been proved to be a multifunctional protein associated with cell proliferation, differentiation, transmembrane transportation and stress response. The expression of NDRG2 is transcriptionally regulated by Myc, TNFαIGF-1, hypoxia, DNA damage and many hormones including dexamethasone, insulin, androgens and aldosterone. In this study, we analyzed the promoter region flanking 5’ of NDRG2 and found a potential ERE (estrogen response element). Moreover, we revealed that estrogencan up-regulate the expression of NDRG2 inboth dose and time-dependent manners. In addition, we demonstrated that ERβ but not ERα, bound specifically to the ERE at position of -1455 to -1131 bp of NDRG2 promoter and trans-activated NDRG2 promoter. These data mean that estrogen as an important circulating hormone also plays a regulative role in NDRG2 expression. In our previous studies we found that NDRG2 interacts with β1-subunit of Na+/K+-ATPase and is involved in estrogen-mediated Na+ and Cl− transport in some epithelial cells. Therefore, characterization of the novel estrogen/NDRG2/Na+/K+-ATPase β1 regulation pathway will broaden the understanding of the regulatory role of estrogen on Na+/K+-ATPase and distribution of this pathway may potentially provide a basis for the intervention of isohydria and internal environment homeostasis in some pathological conditions.
Sai Vishnu Priya
Centre for Cellular & Molecular Biology, India
Title: Expression analysis of candidate genes present in the QTL regions for both iron and zinc in the F7 RILs of Madhukarx Swarna
Time : 15:30-15:50
Biography:
Sai Vishnu Priya has completed her PhD in 2004 from Sri Venkateswara University; worked as post doctoral fellow at Directorate of Rice Research, and visiting scientist at International Crops Research Institute for Semi-Arid Tropics (ICRISAT), Hyderabad. Now, she is working as Project Investigator (PI) (DST, WOS A) at Centre for Cellular and Molecular Biology, Hyderabad.
Abstract:
Micronutrients are necessary for both plant and human survival. Rice is the staple food for fifty percent of the world’s population. However, the polished grain, also known as white rice, contains nutritionally insufficient concentrations of iron (Fe) and Zinc (Zn) to meet the daily requirements in diet leading to adverse effect on human health. Identification of candidate genes in major QTLs and their transcriptome analysis will be useful in gene discovery for biofortification. Genome- wide maps showed 14 QTLs for iron and zinc concentration in unpolished rice grains of F7 recombinant inbred lines from Madhukar x Swarna. Five candidate genes (OsNAS3, OsNRAMP1, OsHMA, OsAPRT and OsZIP8) which encode for both Fe and Zn and underlie four major QTLs on chromosomes 7 and 12 were used for expression analysis. Three leaf stage (32- days old) greenhouse grown plants showed ≥2.15 & ≥1.8 fold expressions of OsNAS3 and OsHMA respectively compared to Madhukar. When 7- days old seedlings were transferred to hoagland solution supplemented with Fe (0.18 mM) and Zn (0.26 mM) and grown for 25- days, all high iron zinc lines (HL) and 75 percent of low iron zinc lines (LL) showed increased expression of OsZIP8 (≥2.2 fold) while other genes were down regulated. Omission of Fe and Zn resulted in stunted growth with reduction in chlorophyll, primary root length and number of roots. In omission of Fe and Zn, showed high transcript levels in all HLs and one LL-270(M) with OsNRAMP1, OsHMA compare to sufficient conditions. Sequence analysis of OsHMA in HL and LL showed variations at 32nd and 37th positions where valine is replaced by alanine and arginine with cystein respectively in 75 percent LL.
- Track 8: Functional Impact of Non-Coding RNA (ncRNA), Track 9: Cancer Genomics: Integrative And Computational Approaches, Track 12: Transcriptional Regulation and Transcriptional Attenuation
Location: Continental Ball Room 5 & 7
Chair
Harry Jarrett
University of Texas San Antonio, USA
Co-Chair
Ding-Gan Liu
Chinese Academy of Sciences, China
Session Introduction
Harry Jarrett
University of Texas San Antonio, USA
Title: New method for analyzing transcription factors and other proteins involved in transcription using MALDI-TOF mass spectrometry
Time : 16:05-16:25
Biography:
Jarrett received his Ph.D. from the Univ. North Carolina-Chapel Hill in 1976. After postdoctoral fellowships at the Mayo Clinic, UCSD, and U.GA , he began an assistant professorship at IN U-Purdue U at Indianapolis. After promotion he joined the faculty at the U. TN Health Science Center where he was promoted to professor and remained for 18 years. In 2006, he joined the faculty at U. TX San Antonio as the Lutcher Brown Distinguished Professor of Biochemistry where he remains. His research has involved the purification of transcription factors and the RNA pol 2 transcription complex. His discoveries have included coupling methods which led to the first DNA-silica HPLC columns and developed both oligonucleotide trapping and promoter trapping to purify transcription proteins for mass spectrometry characterization. Most recently, this research has involved coating MALDI plates to direct proteomic discovery to DNA-binding proteins for a new method called T3.
Abstract:
MALDI plates coated with polyvinylpyrrolidone do not bind protein but avidly bind DNA and DNA-protein complexes. The DNA sequence determines which proteins bind. When DNA, either oligonucleotides or longer DNAs such as core promoter sequences, is mixed with nuclear extract and spotted on the plate, DNA-protein complexes are bound while other proteins can be simply washed away. The samples are then digested with trypsin and matrix is added on the plate and the plate is then ready for mass spectrometry analysis. Highest signal-to-noise is obtained with core promoters (in this case a 300 bp human telomerase (hTERT) promoter) than with 20-mer duplex oligonucleotides but usable data is obtained with either. Using overlapping oligonucleotides representing the entire hTERT promoter, binding sites for TCF23 (an E-box binding protein), SP1, and AP2 were all localized to specific sequence regions. When the intact promoter is used, SP1 and AP2 are also found but instead of TCF23, USF2 is found. This suggests that USF2 binds to the E-boxes in the promoter context. Other components of the RNA polymerase 2 transcription complex such as TF2H were also characterized. The method allows as many as 384 samples to be analyzed on a single plate in a matter of three days and thus the method is capable of high-throughput analysis of DNA-binding proteins.
Ding-Gan Liu
Chinese Academy of Sciences, China
Title: Independent 3’untranslated region RNA: A novel non-coding regulator RNA
Time : 16:25-16:45
Biography:
Ding-Gan Liu has completed his M.S. from the Shanghai Institute of Biochemistry, Chinese Academy of Sciences (CAS) and gradually upgraded to research associate, associate professor and full professor in that Institute (now the Institute of Biochemistry and Cell Biology, Shanghai institutes for Biological Sciences, CAS). During 1980's he was a visiting scientist in the Institute for Physical and Chemical Research (RIKEN), Tokyo, Japan, and in the Tsukuba Life Science Center, RIKEN, Japan. He has been the first author, corresponding author or co-author of more than 50 research papers in Chinese and international scientific journals, such as Nucleic Acids Res, PloS One, BBRC, DNA Cell Biol, and Nature etc.
Abstract:
Classically, the 3’untranslated region (3’UTR) is that region in eukaryotic protein-coding genes from the translation termination codon to the polyA signal. It is transcribed as an integral part of the mRNA encoded by the gene. However, there exists another kind of RNA, which consists of the 3’UTR alone, without all other elements in mRNA such as 5’UTR and coding region. The importance of independent 3’UTR RNA (referred as I3’UTR) was prompted by results of artificially introducing such RNA species into malignant mammalian cells. Since 1991, we found that the middle part of the 3’UTR of the human nuclear factor for interleukin-6 (NF-IL6) or C/EBP gene exerted tumor suppression effect in vivo. Our subsequent studies showed that transfection of C/EBP 3’UTR led to down-regulation of several genes favorable for malignancy and to up-regulation of some genes favorable for phenotypic reversion. Also, it was shown that the sequences near the termini of the C/EBP 3’UTR were important for its tumor suppression activity. Then, the C/EBP 3’UTR was found to directly inhibit the phosphorylation activity of protein kinase CPKC in SMMC-7721, a hepatocarcinoma cell line. Recently, an AU-rich region in the C/EBP 3’UTR was found also to be responsible for its tumor suppression. Recently we have also found evidence that the independent C/EBP 3’UTR RNA is actually exists in human tissues, such as fetal liver and heart, pregnant uterus, senescent fibroblasts etc. Through 1990’s to 2000’s, world scientists found several 3’UTR RNAs that functioned as artificial independent RNAs in cancer cells and resulted in tumor suppression. Interestingly, majority of genes for these RNAs have promoter-like structures in their 3’UTR regions, although the existence of their transcribed products as independent 3’UTR RNAs is still to be confirmed. Our studies indicate that the independent 3’UTR RNA is a novel non-coding RNA species whose function should be the regulation not of the expression of their original mRNA, but of some essential life activities of the cell as a whole.
Yosef Yarden
Weizmann Institute of Science, Israel
Title: Transcription-based chronobiology of receptor tyrosine kinases: Relevance to cancer progression
Time : 16:45-17:05
Biography:
Yosef Yarden completed his PhD from The Weizmann Institute of Science; Rehovot, Israel in the year 1985. Later he went for his postdocs and got appointed as a Senior Scientist in Department of Immunology in The Weizmann Institute in 1988. Currently he is working as a Professor in Department of Molecular Cell Biology in the same institute. He has many awards on his name mentioning some the EMET prize in Biochemistry in 2007 and the MERIT award of the U.S. National Cancer Institute in 2005.
Abstract:
Tumor-specific combinations of oncogenic mutations often free cancer cells from their reliance on growth factors. One important example comprises the epidermal growth factor receptor (EGFR) and its kin, HER2. In tumors, both EGFR and HER2 frequently display overexpression, internal deletions and point mutations. Accordingly, monoclonal antibodies and kinase inhibitors specific to these receptors have been approved for clinical application. The lecture will introduce efforts to resolve the logic underlying gene expression programs activated by growth factors and relationships to tumor progression. Wave-like induction of defined groups of transcripts follows receptor activation, and a similar pattern is displayed by microRNAs. Interestingly, the earliest event we detect is a concerted downregulation of microRNAs that collectively suppress the earliest wave of up-regulated genes, a group of transcription factors that includes FOS and JUN. Importantly, the waves of mRNAs and microRNAs are reflected in breast and other tumors, implying that growth factors play vital roles in tumor progression. The lecture will focus on two emerging features of gene expression programs, which are initiated by activated receptors for growth factors. The first feature is a seemingly pulsatile mode of signals that regulates cell cycle progression. Accordingly, commitment to S-phase entry depends on a fruitful second pulse of signaling, which removes inhibition by the wild type form of p53. The other feature relates to an apparent diurnal regulation of growth factor signaling. This requires signaling by steroid hormones and entails a crosstalk between nuclear receptors and receptor tyrosine kinases, such as EGFR. The implications of both pulsatile regulation of transcription and its diurnal control will be discussed in the context of tumor progression.
Nancy Amaral Reboucas
University of Sao Paulo, Brazil
Title: Parathyroid hormone regulates Nhe3 gene core promoter: EGR1 and Sp3 might regulate the RNA polymerase II pause?
Time : 17:05-17:25
Biography:
Nancy Amaral Reboucas graduated as a medical doctor in 1976 at Federal University of Goiás, Brazil. She concluded my medical residence in Nephrology in 1979, and my PhD in Human Physiology at the Institute of Biomedical Science, Department of Physiology and Biophysics, University of São Paulo, in 1983. Then, she went on to do post-doctoral fellowship at Yale University for two years, from 1989 to 1991, in the Peter Aronson and Peter Igarashi laboratory, Department of Internal Medicine, section of nephrology. Since then, she coordinates her own laboratory at University of São Paulo, in the Department of Physiology and Biophysics, where she teaches renal physiology and membrane physiology to medical students. Her main line of research is transcriptional and functional regulation of the sodium-hydrogen exchanger NHE3 in renal tubules. She is also responsible for a course on Molecular Biology since 1998 that is open to the community. The purpose of this course is introduce graduate students and medical and biomedical professionals to the Molecular Biology of the cell and to the fundamental methods used in a Molecular Biology laboratory.
Abstract:
The main Na+ reabsorption mechanism in the renal proximal tubules is the Na+/H+ exchanger 3 (NHE3) which is acutely and chronically down regulated by parathyroid hormone (PTH). In rats, continuous administration of PTH to induce hyperparathyroidism reduces the expression of NHE3 both at RNA and protein level with clear decrease of NHE3 at the apical membrane of proximal tubules. Analysis with reporter gene of the rat Nhe3 gene promoter in Opossum Kidney Proximal Tubule (OKP) cells indicated inhibition of transcription by PTH associated to a decrease in NHE3-mRNA stability. We demonstrated that PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways changed PTH from an inhibitor into an activator of promoter activity as did point mutations in the EGR1, Sp1 and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1-mRNA and of some Sp3-protein isoforms. Electrophoretic mobility shift assay showed a super shift of the -61 to -42bp probe with an anti-EGR1 antibody in PTH-treated cells suggesting the EGR1 binding is relevant for the inhibitory activity of PTH. Our results suggest that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression to EGR1 binding to the proximal and core promoters and to PKA and JAK/STAT pathway activation. The higher expression of the long isoform (Sp3-li, 90 kDa) and of the short isoform (Sp3-si3) of Sp3 in addition to higher sumoylation of one of the short isoforms of Sp3 induced by PTH could also be related to repression of Nhe3gene expression. As Sp3 was reported to promote RNA polymerase II pause by recruiting phosphatases to the p21CIP1 gene promoter, we hypothesize the PTH might increase RNA Pol II pause at the Nhe3 gene promoter.
Wei Zhang
The University of Texas M.D. Anderson Cancer Center, USA
Title: Interrogating Cancer Transcriptome reprogramming
Biography:
Wei Zhang is a Professor in Pathology and Cancer Biology in the Department of Pathology and the director of the Cancer Genomics Laboratory at MD Anderson Cancer Center. Dr. Zhang has published more than close to 300 peer-reviewed papers and 21 book chapters. Dr. Zhang co-edited two books (Statistical and Computational Approaches to Genomics, 1st and 2nd edition, and Genomic and Molecular Neuro-Oncology) and co-authored one book (Microarray Quality Control). Dr. Zhang is a co-director of one of seven Genome Data Analysis Centers (GDAC) under the Cancer Genome Atlas (TCGA) project funded by NCI.
Abstract:
Recent high throughput interrogations of cancer landscape through programs such as The Cancer Genome Atlas (TCGA) have gained deeper understanding of cancer as a disease of heterogeneity of different stages of cell fate evolution and dedifferentiation. Epithelial tumors often exhibit mesenchymal features as a result of epithelial to mesenchymal transition (EMT), a process that can be driven by both genetic and epigenetic alterations in a form of network regulation. In ovarian cancer, loss of epithelial features and gain of a mesenchymal phenotype is associated with more aggressive tumor features. We performed an integrated genomic analysis, which revealed a miRNA-regulatory network that further defined a robust integrated mesenchymal subtype associated with poor overall survival in 459 cases of serous ovarian cancer from TCGA and 560 cases from independent cohorts. Eight key miRNAs, including miR-506, miR-141, and miR-200a, were predicted to regulate 89% of the targets in this network. Follow-up functional experiments illustrate that miR-506 inhibited EMT by targeting SNAI2, a transcriptional repressor of E-cadherin, as well as mesenchymal regulators vimentin and N-cadherin. MiR-506 also induced senescence by blocking CDK4/6-FoxM1 axis. We used our well-characterized nanoparticle platforms for systemic delivery of miR-506 in orthotopic OvCa mouse models and found such delivery to be highly effective for reduced tumor growth. Interestingly, mesenchymal tumors such as leiomyosarcoma and synovial sarcoma often exhibit epithelial features in a process of mesenchymal to epithelial transition (MET). MET in sarcoma is associated with improved prognosis. Therefore, reprogramming is an intrinsic hallmark of cancer. Understanding of this reprogramming promises to lead to intervention that program cancer into an attractor stage that is more similar to normal cells or a stage that leads to irreversible cell death.