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Juan Carlos Polanco,
Mirabelle S H Ho,
Bei Wang,
Qi Zhou,
Ernst Wolvetang,
Elizabeth Mason,
Christine A Wells,
Gabriel Kolle, Sean M Grimmond,
Ivan Bertoncello,
Carmel O'Brien,
Andrew L Laslett
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ABSTRACT: Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal non-integrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally-derived hiPSC lines re-acquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify "unsafe" hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these "unsafe" hiPSC lines should be interpreted carefully.
Stem Cells 06/2013; · 7.78 Impact Factor
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Ying Cao,
Luke Hoeppner,
Steven Bach,
Guangqi E,
Yan Guo,
Enfeng Wang,
Jianmin Wu,
Mark J Cowley,
David K Chang,
Nicola Waddell, Sean M Grimmond,
Andrew V Biankin,
Roger J Daly,
Xiaohui Zheng,
Debabrata Mukhopadhyay
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ABSTRACT: Metastasis, the leading cause of cancer death, requires tumor cell intravasation, migration through the bloodstream, arrest within capillaries, and extravasation to invade distant tissues. Few mechanistic details have been reported thus far regarding the extravasation process or re-entry of circulating tumor cells at metastatic sites. Here, we demonstrate that neuropilin-2 (NRP-2), a multi-functional non-kinase receptor for semaphorins, vascular endothelial growth factor (VEGF), and other growth factors, expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular extravasation and metastasis in zebrafish and murine xenograft models of clear cell renal cell carcinoma (RCC) and pancreatic adenocarcinoma. In tissue from RCC patients, NRP-2 expression is positively correlated with tumor grade and highest in metastatic tumors. In a prospectively acquired cohort of patients with pancreatic cancer, high NRP-2 expression co-segregated with poor prognosis. Through biochemical approaches as well as Atomic Force Microscopy (AFM), we describe a unique mechanism through which NRP-2 expressed on cancer cells interacts with α5 integrin on endothelial cells to mediate vascular adhesion and extravasation. Taken together, our studies reveal a clinically significant role of NRP-2 in cancer cell extravasation and promotion of metastasis.
Cancer Research 05/2013; · 7.86 Impact Factor
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ABSTRACT: Pancreatic cancer is the fourth leading cause of cancer death in our society, with a mortality that virtually parallels its incidence, a median survival of <12 months even with maximal therapy, and a 5-year survival rate of <5 %. The diversity of clinical outcomes and the molecular heterogeneity of histopathologically similar cancer types, incomplete knowledge of the genomic aberrations that drive carcinogenesis and the lack of therapeutics that specifically target most known genomic aberrations necessitates large-scale detailed analysis of cancer genomes to identify novel potential therapeutic strategies. As part of the International Cancer Genome Consortium (ICGC), the Australian Pancreatic Cancer Genome Initiative (APGI) used exomic sequencing and copy number analysis to define genomic aberrations that characterize a large, clinically focused, prospectively accrued cohort of patients with pancreatic cancer. The cohort consisted of early (clinical stages I and II) non-pre-treated patients with pancreatic ductal adenocarcinoma who underwent operative resection with curative intent. We devised approaches to adjust for low epithelial content in primary tumours and to define the genomic landscape of pancreatic cancer to identify novel candidate driver genes and mechanisms. We aim to develop stratified, molecular phenotype-guided therapeutic strategies using existing therapeutics that are either rescued, repurposed, in development, or are known to be effective in an undefined subgroup of PC patients. These are then tested in primary patient-derived xenografts and cell lines from the above deeply characterized cohort. In addition, we return information to treating clinicians that influences patient care and are launching a clinical trial called IMPaCT (Individualized Molecular Pancreatic Cancer Therapy). This umbrella design trial randomizes patients with metastatic disease to either standard first-line therapy with gemcitabine, or a molecular phenotype-guided approach using next-generation sequencing strategies to screen for actionable mutations defined through the ICGC effort.
Journal of hepato-biliary-pancreatic sciences. 05/2013;
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Joanna Rakoczy,
Selene L Fernandez-Valverde,
Evgeny A Glazov,
Elanor N Wainwright,
Tempei Sato,
Shuji Takada,
Alexander N Combes,
Darren J Korbie,
David Miller, Sean M Grimmond,
Melissa H Little,
Hiroshi Asahara,
John S Mattick,
Ryan J Taft,
Dagmar Wilhelm
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ABSTRACT: MicroRNAs (miRNAs) have been shown to play key regulatory roles in a range of biological processes, including cell differentiation and development. To identify miRNAs that participate in gonad differentiation, a fundamental and tightly regulated developmental process, we examined miRNA expression profiles at the time of sex determination and during the early fetal differentiation of mouse testes and ovaries using high-throughput sequencing. We identified several miRNAs that were expressed in a sexually dimorphic pattern, including several members of the let-7 family, miR-378 and miR-140-3p. We focused our analysis on the most highly expressed sexually dimorphic miRNA, miR-140-3p, and found that both miR-140-3p and its more lowly expressed counterpart, the previously annotated guide strand, miR-140-5p, are testis-enriched and expressed in testis cords. Analysis of the miR-140-5p/miR-140-3p null mouse revealed a significant increase in the number of Leydig cells in the developing XY gonad, strongly suggesting an important role for miR-140-5p/miR-140-3p in testis differentiation in mouse.
Biology of Reproduction 04/2013; · 4.01 Impact Factor
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John M Atack,
Yogitha N Srikhanta,
Karrera Y Djoko,
Jessica P Welch,
Norain H M Hasri,
Christopher T Steichen,
Rachel N Vanden Hoven, Sean M Grimmond,
Dk Seti Maimonah Pg Othman,
Ulrike Kappler,
Michael A Apicella,
Michael P Jennings,
Jennifer L Edwards,
Alastair G McEwan
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ABSTRACT: NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of α-Proteobacteria. Phylogenetic analysis of the response regulator, NtrX, showed that this two-component system is extensively distributed across the bacterial domain and it is present in a variety of β-Proteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in a N. gonorrhoeae ntrX mutant compared to the isogenic wild-type strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA) and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild-type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by live-dead staining. Analyses of an ntrX mutant in a representative α-Proteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to the wild-type strain, SB1003. Taken together, these data provide evidence that the NtrYX two component-system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.
Journal of bacteriology 04/2013; · 3.94 Impact Factor
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John M. Atack,
Yogitha N. Srikhanta,
Karrera Y. Djoko,
Jessica P. Welch,
Norain H. M. Hasri,
Christopher T. Steichen,
Rachel N. Vanden Hoven, Sean M. Grimmond,
Dk Seti Maimonah Pg Othman,
Ulrike Kappler,
Michael A. Apicella,
Michael P. Jennings,
Jennifer L. Edwards,
Alastair G. McEwan
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ABSTRACT: Large-scale cancer genome studies are unveiling significant complexity and heterogeneity even in histopathologically indistinguishable cancers. Differentiating 'driver' mutations that are functionally relevant from 'passenger' mutations is a major challenge in cancer genomics. While recurrent mutations in a gene provides supporting evidence of 'driver' status, novel computational methods and model systems are greatly improving our ability to identify genes important in carcinogenesis. Reimand and Bader have recently shown that driver gene discovery in discrete gene classes (in this case the kinome) is possible across multiple cancer types and has the potential to yield new druggable targets and clinically relevant leads.
Genome Medicine 02/2013; 5(2):19.
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Keerthana Krishnan,
Anita L Steptoe,
Hilary C Martin,
Shivangi Wani,
Katia Nones,
Nic Waddell,
Mythily Mariasegaram,
Peter T Simpson,
Sunil R Lakhani,
Brian Gabrielli,
Alexander Vlassov,
Nicole Cloonan, Sean M Grimmond
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ABSTRACT: MicroRNAs are noncoding regulators of gene expression, which act by repressing protein translation and/or degrading mRNA. Many have been shown to drive tumorigenesis in cancer, but functional studies to understand their mode of action are typically limited to single-target genes. In this study, we use synthetic biotinylated miRNA to pull down endogenous targets of miR-182-5p. We identified more than 1000 genes as potential targets of miR-182-5p, most of which have a known function in pathways underlying tumor biology. Specifically, functional enrichment analysis identified components of both the DNA damage response pathway and cell cycle to be highly represented in this target cohort. Experimental validation confirmed that miR-182-5p-mediated disruption of the homologous recombination (HR) pathway is a consequence of its ability to target multiple components in that pathway. Although there is a strong enrichment for the cell cycle ontology, we do not see primary proliferative defects as a consequence of miR-182-5p overexpression. We highlight targets that could be responsible for miR-182-5p-mediated disruption of other biological processes attributed in the literature so far. Finally, we show that miR-182-5p is highly expressed in a panel of human breast cancer samples, highlighting its role as a potential oncomir in breast cancer.
RNA 12/2012; · 5.09 Impact Factor
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Andrew V Biankin,
Nicola Waddell,
Karin S Kassahn,
Marie-Claude Gingras,
Lakshmi B Muthuswamy,
Amber L Johns,
David K Miller,
Peter J Wilson,
Ann-Marie Patch,
Jianmin Wu, [......],
David A Largaespada,
Lodewyk F A Wessels,
Alistair G Rust,
Lincoln D Stein,
David A Tuveson,
Neal G Copeland,
Thomas J Hudson,
David A Wheeler,
John D McPherson,
Richard A Gibbs
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ABSTRACT: Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.
Nature 10/2012; · 36.28 Impact Factor
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Andrew V. Biankin,
Nicola Waddell,
Karin S. Kassahn,
Marie-Claude Gingras,
Lakshmi B. Muthuswamy,
Amber L. Johns,
David K. Miller,
Peter J. Wilson,
Ann-Marie Patch,
Jianmin Wu, [......],
Elizabeth A. Musgrove,
Aldo Scarpa,
James R. Eshleman,
Thomas J. Hudson,
Robert L. Sutherland,
David A. Wheeler,
John V. Pearson,
John D. McPherson,
Richard A. Gibbs, Sean M. Grimmond
Nature 10/2012; advance online publication. · 36.28 Impact Factor
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Carole M Tactacan,
David K Chang,
Mark J Cowley,
Emily S Humphrey,
Jianmin Wu,
Anthony J Gill,
Angela Chou,
Katia Nones, Sean M Grimmond,
Robert L Sutherland,
Andrew V Biankin,
Roger J Daly
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ABSTRACT: BACKGROUND: The receptor tyrosine kinase RON exhibits increased expression during pancreatic cancer progression and promotes migration, invasion and gemcitabine resistance of pancreatic cancer cells in experimental models. However, the prognostic significance of RON expression in pancreatic cancer is unknown. METHODS: RON expression was characterized in several large cohorts, including a prospective study, totaling 492 pancreatic cancer patients and relationships with patient outcome and clinico-pathologic variables were assessed. RESULTS: RON expression was associated with outcome in a training set, but this was not recapitulated in the validation set, nor was there any association with therapeutic responsiveness in the validation set or the prospective study. CONCLUSIONS: Although RON is implicated in pancreatic cancer progression in experimental models, and may constitute a therapeutic target, RON expression is not associated with prognosis or therapeutic responsiveness in resected pancreatic cancer.
BMC Cancer 09/2012; 12(1):395. · 3.01 Impact Factor
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Jing Yu,
M Todd Valerius,
Mary Duah,
Karl Staser,
Jennifer K Hansard,
Jin-Jin Guo,
Jill McMahon,
Joe Vaughan,
Diane Faria,
Kylie Georgas, [......],
Philip Machanick,
Paul A Gray,
Alexander van Oudenaarden,
David H Rowitch,
Charles D Stiles,
Qiufu Ma, Sean M Grimmond,
Timothy L Bailey,
Melissa H Little,
Andrew P McMahon
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ABSTRACT: Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.
Development 05/2012; 139(10):1863-73. · 6.60 Impact Factor
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Karen M Mann,
Jerrold M Ward,
Christopher Chin Kuan Yew,
Anne Kovochich,
David W Dawson,
Michael A Black,
Benjamin T Brett,
Todd E Sheetz,
Adam J Dupuy,
David K Chang,
Andrew V Biankin,
Nicola Waddell,
Karin S Kassahn, Sean M Grimmond,
Alistair G Rust,
David J Adams,
Nancy A Jenkins,
Neal G Copeland
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ABSTRACT: Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma.
Proceedings of the National Academy of Sciences 03/2012; 109(16):5934-41. · 9.68 Impact Factor
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Rebecca A Pelekanos,
Joan Li,
Milena Gongora,
Vashe Chandrakanthan,
Janelle Scown,
Norseha Suhaimi,
Gary Brooke,
Melinda E Christensen,
Tram Doan,
Alison M Rice,
Geoffrey W Osborne, Sean M Grimmond,
Richard P Harvey,
Kerry Atkinson,
Melissa H Little
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ABSTRACT: Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.
Stem cell research 01/2012; 8(1):58-73. · 3.39 Impact Factor
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Sarah Song,
Katia Nones,
David Miller,
Ivon Harliwong,
Karin S Kassahn,
Mark Pinese,
Marina Pajic,
Anthony J Gill,
Amber L Johns,
Matthew Anderson, [......],
Qinying Xu,
Felicity Newell,
Mark J Cowley,
Jianmin Wu,
Peter Wilson,
Lynn Fink,
Andrew V Biankin,
Nic Waddell, Sean M Grimmond,
John V Pearson
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ABSTRACT: Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH) in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 ([Formula: see text]-value = 0.0001) between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 ([Formula: see text]-value [Formula: see text] 2.2e-16) between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 ([Formula: see text]-value = 0.004) between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.
PLoS ONE 01/2012; 7(9):e45835. · 4.09 Impact Factor
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Huijun Chen,
James S Palmer,
Rathi D Thiagarajan,
Marcel E Dinger,
Emmanuelle Lesieur,
Hansheng Chiu,
Alexandra Schulz,
Cassy Spiller, Sean M Grimmond,
Melissa H Little,
Peter Koopman,
Dagmar Wilhelm
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ABSTRACT: In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process.
PLoS ONE 01/2012; 7(7):e41683. · 4.09 Impact Factor
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Nicole Cloonan,
Shivangi Wani,
Qinying Xu,
Jian Gu,
Kristi Lea,
Sheila Heater,
Catalin Barbacioru,
Anita L Steptoe,
Hilary C Martin,
Ehsan Nourbakhsh, [......],
David L Wood,
Karin S Kassahn,
Nic Waddell,
Jill Shepherd,
Clarence Lee,
Jeff Ichikawa,
Kevin McKernan,
Kelli Bramlett,
Scott Kuersten, Sean M Grimmond
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ABSTRACT: Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules.
To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs.
Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.
Genome biology 12/2011; 12(12):R126. · 6.63 Impact Factor
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Gabriel Kolle,
Jill L Shepherd,
Brooke Gardiner,
Karin S Kassahn,
Nicole Cloonan,
David L A Wood,
Ehsan Nourbakhsh,
Darrin F Taylor,
Shivangi Wani,
Hun S Chy,
Qi Zhou,
Kevin McKernan,
Scott Kuersten,
Andrew L Laslett, Sean M Grimmond
[show abstract]
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ABSTRACT: Recent RNA-sequencing studies have shown remarkable complexity in the mammalian transcriptome. The ultimate impact of this complexity on the predicted proteomic output is less well defined. We have undertaken strand-specific RNA sequencing of multiple cellular RNA fractions (>20 Gb) to uncover the transcriptional complexity of human embryonic stem cells (hESCs). We have shown that human embryonic stem (ES) cells display a high degree of transcriptional diversity, with more than half of active genes generating RNAs that differ from conventional gene models. We found evidence that more than 1000 genes express long 5' and/or extended 3'UTRs, which was confirmed by "virtual Northern" analysis. Exhaustive sequencing of the membrane-polysome and cytosolic/untranslated fractions of hESCs was used to identify RNAs encoding peptides destined for secretion and the extracellular space and to demonstrate preferential selection of transcription complexity for translation in vitro. The impact of this newly defined complexity on known gene-centric network models such as the Plurinet and the cell surface signaling machinery in human ES cells revealed a significant expansion of known transcript isoforms at play, many predicting possible alternative functions based on sequence alterations within key functional domains.
Genome Research 12/2011; 21(12):2014-25. · 13.61 Impact Factor
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ABSTRACT: The Protein Interaction Network Analysis (PINA) platform is a comprehensive web resource, which includes a database of unified protein-protein interaction data integrated from six manually curated public databases, and a set of built-in tools for network construction, filtering, analysis and visualization. The second version of PINA enhances its utility for studies of protein interactions at a network level, by including multiple collections of interaction modules identified by different clustering approaches from the whole network of protein interactions ('interactome') for six model organisms. All identified modules are fully annotated by enriched Gene Ontology terms, KEGG pathways, Pfam domains and the chemical and genetic perturbations collection from MSigDB. Moreover, a new tool is provided for module enrichment analysis in addition to simple query function. The interactome data are also available on the web site for further bioinformatics analysis. PINA is freely accessible at http://cbg.garvan.unsw.edu.au/pina/.
Nucleic Acids Research 11/2011; 40(Database issue):D862-5. · 8.03 Impact Factor
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Rathi D Thiagarajan,
Nicole Cloonan,
Brooke B Gardiner,
Tim R Mercer,
Gabriel Kolle,
Ehsan Nourbakhsh,
Shivangi Wani,
Dave Tang,
Keerthana Krishnan,
Kylie M Georgas,
Bree A Rumballe,
Han S Chiu,
Jason A Steen,
John S Mattick,
Melissa H Little, Sean M Grimmond
[show abstract]
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ABSTRACT: The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined with systematic in situ screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models.
To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3' UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section in situ hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs.
The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.
BMC Genomics 09/2011; 12:441. · 4.07 Impact Factor
