Deep-transcriptome and ribonome sequencing redefines the molecular networks of pluripotency and the extracellular space in human embryonic stem cells.

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.

RNA-MATE: a recursive mapping strategy for high-throughput RNA-sequencing data.

UNLABELLED: Mapping of next-generation sequencing data derived from RNA samples (RNAseq) presents different genome mapping challenges than data derived from DNA. For example, tags that cross exon-junction boundaries will often not map to a reference genome, and the strand specificity of the data needs to be retained. Here we present RNA-MATE, a computational pipeline based on a recursive mapping strategy for placing strand specific RNAseq data onto a reference genome. Maximizing the mappable tags can provide significant savings in the cost of sequencing experiments. This pipeline provides an automatic and integrated way to align color-space sequencing data, collate this information and generate files for examining gene-expression data in a genomic context. AVAILABILITY: Executables, source code, and exon-junction libraries are available from http://grimmond.imb.uq.edu.au/RNA-MATE/

Stem cell transcriptome profiling via massive-scale mRNA sequencing.

We developed a massive-scale RNA sequencing protocol, short quantitative random RNA libraries or SQRL, to survey the complexity, dynamics and sequence content of transcriptomes in a near-complete fashion. This method generates directional, random-primed, linear cDNA libraries that are optimized for next-generation short-tag sequencing. We surveyed the poly(A)(+) transcriptomes of undifferentiated mouse embryonic stem cells (ESCs) and embryoid bodies (EBs) at an unprecedented depth (10 Gb), using the Applied Biosystems SOLiD technology. These libraries capture the genomic landscape of expression, state-specific expression, single-nucleotide polymorphisms (SNPs), the transcriptional activity of repeat elements, and both known and new alternative splicing events. We investigated the impact of transcriptional complexity on current models of key signaling pathways controlling ESC pluripotency and differentiation, highlighting how SQRL can be used to characterize transcriptome content and dynamics in a quantitative and reproducible manner, and suggesting that our understanding of transcriptional complexity is far from complete.

Genome-wide review of transcriptional complexity in mouse protein kinases and phosphatases.

BACKGROUND: Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse http://variant.imb.uq.edu.au. RESULTS: By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys (seven known and 19 novel: Alk, Csf1r, Egfr, Epha1, 3, 5,7 and 10, Ephb1, Flt1, Flt3, Insr, Insrr, Kdr, Met, Ptk7, Ptprc, Ptprd, Ptprg, Ptprl, Ptprn, Ptprn2, Ptpro, Ptprr, Ptprs, and Ptprz1) and 13 transmembrane forms (four known and nine novel: Axl, Bmpr1a, Csf1r, Epha4, 5, 6 and 7, Ntrk2, Ntrk3, Pdgfra, Ptprk, Ptprm, Ptpru). Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms. CONCLUSION: These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways.

PhosphoregDB: the tissue and sub-cellular distribution of mammalian protein kinases and phosphatases.

BACKGROUND: Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse http://phosphoreg.imb.uq.edu.au that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. RESULTS: The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. CONCLUSION: Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered.

Spatial gene expression in the T-stage mouse metanephros.

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.

Differential gene expression in the developing mouse ureter.

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.

Identifying the molecular phenotype of renal progenitor cells.

Although many of the molecular interactions in kidney development are now well understood, the molecules involved in the specification of the metanephric mesenchyme from surrounding intermediate mesoderm and, hence, the formation of the renal progenitor population are poorly characterized. In this study, cDNA microarrays were used to identify genes enriched in the murine embryonic day 10.5 (E10.5) uninduced metanephric mesenchyme, the renal progenitor population, in comparison with more rostral derivatives of the intermediate mesoderm. Microarray data were analyzed using R statistical software to determine accurately genes differentially expressed between these populations. Microarray outliers were biologically verified, and the spatial expression pattern of these genes at E10.5 and subsequent stages of early kidney development was determined by RNA in situ hybridization. This approach identified 21 genes preferentially expressed by the E10.5 metanephric mesenchyme, including Ewing sarcoma homolog, 14-3-3 theta, retinoic acid receptor-alpha, stearoyl-CoA desaturase 2, CD24, and cadherin-11, that may be important in formation of renal progenitor cells. Cell surface proteins such as CD24 and cadherin-11 that were strongly and specifically expressed in the uninduced metanephric mesenchyme and mark the renal progenitor population may prove useful in the purification of renal progenitor cells by FACS. These findings may assist in the isolation and characterization of potential renal stem cells for use in cellular therapies for kidney disease.