The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing.

Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders.

Long noncoding RNAs and the genetics of cancer.

Cancer is a disease of aberrant gene expression. While the genetic causes of cancer have been intensively studied, it is becoming evident that a large proportion of cancer susceptibility cannot be attributed to variation in protein-coding sequences. This is highlighted by genome-wide association studies in cancer that reveal that more than 80% of cancer-associated SNPs occur in noncoding regions of the genome. In this review, we posit that a significant fraction of the genetic aetiology of cancer is exacted by noncoding regulatory sequences, particularly by long noncoding RNAs (lncRNAs). Recent studies indicate that several cancer risk loci are transcribed into lncRNAs and these transcripts play key roles in tumorigenesis. We discuss the epigenetic and other mechanisms through which lncRNAs function and how they contribute to each stage of cancer progression, understanding of which will be crucial for realising new opportunities in cancer diagnosis and treatment. Long noncoding RNAs play important roles in almost every aspect of cell biology from nuclear organisation and epigenetic regulation to post-transcriptional regulation and splicing, and we link these processes to the hallmarks and genetics of cancer. Finally, we highlight recent progress and future potential in the application of lncRNAs as therapeutic targets and diagnostic markers.

An RNA recognition motif in Wilms' tumour protein (WT1) revealed by structural modelling.

The Wilms' tumour suppressor gene 1 (WT1) (1,2) encodes four C2H2 zinc finger-containing proteins (3) critical for normal mammalian urogenital development (4). Mutations in this gene are observed in the childhood kidney cancer, Wilms' tumour (WT) (5). WT1 can bind specific DNA targets within the promoters of many genes (6-9) and both transcriptional repression and activation domains have been identified (10). On this basis, it has been assumed that regulation of transcription is the basis of WT1 tumour suppressor activity. However, subnuclear localization studies have revealed an association between WT1 proteins and 'speckled bodies' within the nucleus. Degradation of nuclear RNA in cells expressing WT1 abolishes this speckled localization and WT1 co-immunoprecipitates with a number of spliceosomal proteins, suggesting that it may also bind to RNA (11). Using structural rather than sequence comparison, we have now identified an evolutionarily conserved N-terminal RNA recognition motif (RRM) in all known WT1 isoforms similar to that in the constitutive splicing factor U1A. Given the association between WT1 mutations and Wilms' tumours, this study, together with other recent findings, may suggest a novel tumour suppression mechanism.

The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.

The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

Introns: evolution and function.

The debate continues on the issue of whether nuclear introns were present in eukaryotic protein-coding genes from the beginning (introns-early) or invaded them later in evolution (introns-late). Recent studies concerning the location of introns with respect to gene and protein structure have been interpreted as providing strong support for both positions, but the weight of argument is clearly moving in favour of the latter. Consistent with this, there is now good evidence that introns can function as transposable elements, and that nuclear introns derived from self-splicing group II introns, which then evolved in partnership with the spliceosome. This was only made possible by the separation of transcription and translation. If introns did colonize eukaryotic genes after their divergence from prokaryotes, the original question as to the evolutionary forces that have seen these sequences flourish in the higher organisms, and their significance in eukaryotic biology, is again thrown open. I suggest that introns, once established in eukaryotic genomes, might have explored new genetic space and acquired functions which provided a positive pressure for their expansion. I further suggest that there are now two types of information produced by eukaryotic genes--mRNA and iRNA--and that this was a critical step in the development of multicellular organisms.

Intergenic disease-associated regions are abundant in novel transcripts.

BACKGROUND: Genotyping of large populations through genome-wide association studies (GWAS) has successfully identified many genomic variants associated with traits or disease risk. Unexpectedly, a large proportion of GWAS single nucleotide polymorphisms (SNPs) and associated haplotype blocks are in intronic and intergenic regions, hindering their functional evaluation. While some of these risk-susceptibility regions encompass cis-regulatory sites, their transcriptional potential has never been systematically explored. RESULTS: To detect rare tissue-specific expression, we employed the transcript-enrichment method CaptureSeq on 21 human tissues to identify 1775 multi-exonic transcripts from 561 intronic and intergenic haploblocks associated with 392 traits and diseases, covering 73.9 Mb (2.2%) of the human genome. We show that a large proportion (85%) of disease-associated haploblocks express novel multi-exonic non-coding transcripts that are tissue-specific and enriched for GWAS SNPs as well as epigenetic markers of active transcription and enhancer activity. Similarly, we captured transcriptomes from 13 melanomas, targeting nine melanoma-associated haploblocks, and characterized 31 novel melanoma-specific transcripts that include fusion proteins, novel exons and non-coding RNAs, one-third of which showed allelically imbalanced expression. CONCLUSIONS: This resource of previously unreported transcripts in disease-associated regions ( http://gwas-captureseq.dingerlab.org ) should provide an important starting point for the translational community in search of novel biomarkers, disease mechanisms, and drug targets.

Escherichia coli gpt as a positive and negative selectable marker in embryonal stem cells.

Transfection of HPRT- L fibroblasts with a plasmid containing two linked selectable markers genes, gpt and neo, regulated by the same eukaryotic control elements, yielded a 6-fold higher transfection frequency on selection for neo than for gpt. Transfection of HPRT- embryonal stem (ES) cells with the same plasmid yielded high levels of transfectants when selected for neo expression with G418, but a level of transfection greater than two orders of magnitude lower was observed when HAT supplemented medium was used to select for gpt expression. Selection for gpt expression in ES cells with medium containing mycophenolic acid and xanthine gave slightly higher frequencies of transfection, but still considerably lower than that for neo selection. In addition, mycophenolic acid exhibited a general cytotoxicity to ES cells with the window between toxicity of this compound to gpt- ES cells and gpt+ ES cells being very narrow. Cells selected with mycophenolic acid and xanthine for expression of gpt remained sensitive to HAT selection. Expression of gpt in a representative ES cell line, selected on mycophenolic acid and xanthine, was verified by Northern analysis and sensitivity to 6-thioguanine. While the level of mRNA expression in this ES cell line was insufficient to support growth via purine salvage when exposed to HAT medium, identical levels of gpt expression in HPRT- L cells, as judged by Northern analysis, allowed for normal growth in HAT medium. This suggests that ES cells place a greater demand on purine nucleotide biosynthesis than L cells. These results are discussed in terms of the use of gpt as a positive and negative selectable marker for gene targeting via homologous recombination in ES cells.

Co-localization of FAM and AF-6, the mammalian homologues of Drosophila faf and canoe, in mouse eye development.

The Drosophila fat facets and canoe genes regulate non-neural cell fate decisions during ommatidium formation. We have shown previously that the FAM (fat facets in mouse) de-ubiquitinating enzyme regulates the function of AF-6, (mammalian canoe homologue), in the MDCK epithelial cell line (Taya et al., 1998. The Ras target AF-6 is a substrate of the fam de-ubiquitinating enzyme. J. Cell Biol. 142, 1053-1062). We report here that the expression of the FAM and AF-6 proteins overlaps extensively in the mouse eye from embryogenesis to maturity, especially in the non-neural epithelia including the retinal pigment epithelium, subcapsular epithelium of the lens and corneal epithelium. Expression is not limited to the epithelia however, as FAM and AF-6 also co-localize during lens fibre development as well as in sub-populations of the neural retina.

FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development.

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta-catenin and AF-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta-catenin such that very little of either protein remained following 72h culture. The residual beta-catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta-catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta-catenin and AF-6 in vivo.

Cloning and expression analysis of a novel mouse gene with sequence similarity to the Drosophila fat facets gene.

The Drosophila fat facets (faf) gene is a ubiquitin-specific protease necessary for the normal development of the eye and of the syncytial stage embryo in the fly. Using a gene trap approach in embryonic stem cells we have isolated a murine gene with extensive sequence similarity to the Drosophila faf gene and called it Fam (fat facets in mouse). The putative mouse protein shows colinearity and a high degree of sequence identity to the Drosophila protein over almost its entire length of 2554 amino acids. The two enzymatic sites characteristic of ubiquitin-specific proteases are very highly conserved between mice and Drosophila and this conservation extends to yeast. Fam is expressed in a complex pattern during postimplantation development. In situ hybridisation detected Fam transcripts in the rapidly expanding cell populations of gastrulating and neurulating embryos, in post-mitotic cells of the CNS as well as in the apoptotic regions between the digits, indicating that it is not associated with a single developmental or cellular event. The strong sequence similarity to faf and the developmentally regulated expression pattern suggest that Fam and the ubiquitin pathway may play a role in determining cell fate in mammals, as has been established for Drosophila.

Accelerating, hyperaccelerating, and decelerating networks.

Many growing networks possess accelerating statistics where the number of links added with each new node is an increasing function of network size so the total number of links increases faster than linearly with network size. In particular, biological networks can display a quadratic growth in regulator number with genome size even while remaining sparsely connected. These features are mutually incompatible in standard treatments of network theory which typically require that every new network node possesses at least one connection. To model sparsely connected networks, we generalize existing approaches and add each new node with a probabilistic number of links to generate either accelerating, hyperaccelerating, or even decelerating network statistics in different regimes. Under preferential attachment for example, slowly accelerating networks display stationary scale-free statistics relatively independent of network size while more rapidly accelerating networks display a transition from scale-free to exponential statistics with network growth. Such transitions explain, for instance, the evolutionary record of single-celled organisms which display strict size and complexity limits.

Inherent size constraints on prokaryote gene networks due to "accelerating" growth.

Networks exhibiting "accelerating" growth have total link numbers growing faster than linearly with network size and either reach a limit or exhibit graduated transitions from nonstationary-to-stationary statistics and from random to scale-free to regular statistics as the network size grows. However, if for any reason the network cannot tolerate such gross structural changes then accelerating networks are constrained to have sizes below some critical value. This is of interest as the regulatory gene networks of single-celled prokaryotes are characterized by an accelerating quadratic growth and are size constrained to be less than about 10,000 genes encoded in DNA sequence of less than about 10 megabases. This paper presents a probabilistic accelerating network model for prokaryotic gene regulation which closely matches observed statistics by employing two classes of network nodes (regulatory and non-regulatory) and directed links whose inbound heads are exponentially distributed over all nodes and whose outbound tails are preferentially attached to regulatory nodes and described by a scale-free distribution. This model explains the observed quadratic growth in regulator number with gene number and predicts an upper prokaryote size limit closely approximating the observed value.

Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa.

Type-4 fimbriae are filamentous polar organelles which are found in a wide variety of pathogenic bacteria. Their biogenesis and function is proving to be extremely complex, involving the expression and coordinate regulation of a large number of genes. Type-4 fimbriae mediate attachment to host epithelial tissues and a form of surface translocation called twitching motility. In Pseudomonas aeruginosa they also appear to function as receptors for fimbrial-dependent bacteriophages. Analysis of mutants defective in fimbrial function has allowed the identification of many of the genes involved in the biogenesis of these organelles. Thus far over 30 genes have been characterized, which fall into two broad categories: those encoding regulatory networks that control the production and function of these fimbriae (and other virulence determinants such as alginate) in response to alterations in environmental conditions; and those encoding proteins involved in export and assembly of these organelles, many of which are similar to proteins involved in protein secretion and DNA uptake. These systems all appear to be closely related and to function in the assembly of surface-associated protein complexes that have been adapted to different biological functions.

Escherichia coli contains a set of genes homologous to those involved in protein secretion, DNA uptake and the assembly of type-4 fimbriae in other bacteria.

A specialised system involved in a diverse array of functions, including the biogenesis of fimbriae, protein secretion and DNA uptake, has recently been found to be widespread in the eubacteria. These systems have in common several sets of related genes, including those encoding proteins containing leader sequences homologous to that of the type-4 fimbrial subunit (prepilin), a prepilin-type leader peptidase, a cytoplasmic nucleotide-binding protein, and other proteins located in the inner and outer membranes [Hobbs, M. and Mattick, J.S., Mol Microbiol. 10 (1993) 233-243]. Here, we show that Escherichia coli contains at least nine homologs of this system, and present complete sequence data for five of the genes involved (ppdD. hopB, hopC, hopD and pshM), as well as for an adjacent gene (nadC), which encodes quinolic acid phosphoribosyltransferase. Insertional mutagenesis of hopB and hopD failed to reveal any obvious effects on cell viability, morphogenesis of M13 phage, conjugative transfer of the F plasmid, or protein secretion.

Sequencing and expression of the aroA gene from Dichelobacter nodosus.

The aroA locus of the Gram- pathogen Dichelobacter nodosus, which encodes 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has been sequenced and expressed in Escherichia coli. The gene is located on a 1.48-kb DraI-HindIII fragment located directly upstream and in opposite transcriptional orientation to the gene encoding the fimbrial structural subunit. The deduced open reading frame is 1329 nucleotides in length, which encodes a protein of 443 amino acids (aa) with a calculated M(r) of 47,413, which was visualized in E. coli minicells, under the control of its native promoter. This derived aa sequence displays significant similarities with the sequences of the aroA gene products from a variety of microorganisms.

Construction of improved vectors for protein production in Pseudomonas aeruginosa.

We report the construction of two cloning vectors that are based on the Pseudomonas-Escherichia shuttle vector, pUCP19. The new vectors, pUCPKS and pUCPSK, contain a significantly expanded multiple cloning site (MCS) with an adjacent T7 promoter sequence. In conjunction with specifically engineered host strains encoding an inducible T7 RNA polymerase, these vectors allow the controlled production of plasmid-encoded proteins in both Escherichia coli and Pseudomonas aeruginosa to analyse the spectrum of products encoded by cloned segments of DNA. The usefulness of these vectors was demonstrated by expressing the chloramphenicol acetyltransferase (CAT)-encoding gene.

Identification of a gene, pilF, required for type 4 fimbrial biogenesis and twitching motility in Pseudomonas aeruginosa.

Many bacterial pathogens produce a class of surface structures called type 4 fimbriae. In Pseudomonas aeruginosa these fimbriae are responsible for adhesion and translocation across host epithelial surfaces. We have identified a novel gene involved in the complex process of type 4 fimbrial biogenesis. This gene, termed pilF, is located on SpeI fragment S at 30 min on the P. aeruginosa genomic map, which is the sixth region on the chromosome shown to contain a fimbrial-associated gene. The PilF protein has a predicted M(r) of 22402, and together with a highly homologous upstream ORF shares a chromosomal arrangement similar to that found in Haemophilus influenzae. A pilF mutant is blocked in the export/assembly of the fimbrial subunit PilA, and accumulates this protein in the membrane fraction. Complementation studies indicate that the cloned pilF gene is able to restore the expression of surface fimbriae, twitching motility and susceptibility to fimbrial-specific bacteriophage.

The molecular genetics of type-4 fimbriae in Pseudomonas aeruginosa--a review.

Type-4 fimbriae (or pili) are filaments found at the poles of a wide range of bacterial pathogens, including Neisseria gonorrhoeae, Moraxella bovis, Dichelobacter nodosus and Pseudomonas aeruginosa. They are composed of a small subunit which is highly conserved among different species and appear to mediate adhesion and translocation across epithelial surfaces via a phenomenon termed "twitching motility'. These fimbriae are key host colonisation factors and important protective antigens. We have analysed the genetics and biosynthesis of type-4 fimbriae in P. aeruginosa, which is an opportunistic pathogen of compromised individuals, including those suffering cystic fibrosis, AIDS or burns. A library of P. aeruginosa transposon mutants was constructed which exhibited loss of twitching motility, as determined by altered colony morphology. Analysis of these mutants, and of similar collections by other groups, have revealed that there are at least 22 genes involved in type-4 fimbrial assembly and function. A large number (pilA, B, C, D, E, M, N, O, P, Q, T, U, V and Z) appear to be involved in the biogenesis of the fimbriae and to represent a subset of a supersystem involved in the assembly of surface-associated protein complexes. Homologs of at least some of these genes have subsequently been identified in other type-4 fimbriate bacteria. In P. aeruginosa, the system is also regulated via two signal transduction pathways-a classic sensor-regulator system (encoded by pilS, pilR and rpoN) which controls transcription of the fimbrial subunit, presumably in response to host cues, and a chemotactic system (encoded by pilG, H, I, J, K and L) which may be involved in the directional or rate control of twitching motility in response to local environmental variables.

Functional expression of heterologous type 4 fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are surface organelles produced by a wide range of bacterial pathogens. In Pseudomonas aeruginosa they are associated with a form of surface translocation known as twitching motility and have also been implicated as the receptor for a number of fimbrial-specific bacteriophages. The infrastructural machinery required for type 4 fimbrial biogenesis appears to be conserved as heterologous subunits from other species can be expressed in P. aeruginosa. All of these studies have, until now, been performed in non-functional Pseudomonas host strains which lack twitching motility. We have constructed isogenic mutants of two commonly studied wild-type P. aeruginosa strains, PAK and PAO1, by replacing the entire pilA gene which encodes the fimbrial subunit. Fimbrial expression and twitching motility were restored by complementation in trans with either the homologous or heterologous subunits from these strains, as well as that from another type 4 fimbriate species, Dichelobacter nodosus. The expression of different subunits allowed us to investigate the precise role that the individual subunit proteins contribute to bacteriophage infection by several fimbrial-specific bacteriophages. Sensitivity to bacteriophages B3cts and D3112cts was restored by the expression of any fimbrial subunit in both PAO1 and PAK cells, indicating that infection by these bacteriophages is fimbrial dependent but not fimbrial specific. In contrast, while sensitivity to the PAK-specific bacteriophage PO4 was restored by the expression of any fimbrial subunit in PAK cells, this did not occur in PAO1 cells except when expressing the PAK subunit. In all cases, the presence of fimbriae was absolutely required to allow a productive bacteriophage infection to occur.