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Background: RNA modifications play central roles in cellular fate and differentiation. However, the machinery responsible for placing, removing, and recognizing more than 170 RNA modifications remains largely uncharacterized and poorly annotated, and we currently lack integrative studies that identify which RNA modification-related proteins (RMPs) may be dysregulated in each cancer type. Results: Here, we perform a comprehensive annotation and evolutionary analysis of human RMPs, as well as an integrative analysis of their expression patterns across 32 tissues, 10 species, and 13,358 paired tumor-normal human samples. Our analysis reveals an unanticipated heterogeneity of RMP expression patterns across mammalian tissues, with a vast proportion of duplicated enzymes displaying testis-specific expression, suggesting a key role for RNA modifications in sperm formation and possibly intergenerational inheritance. We uncover many RMPs that are dysregulated in various types of cancer, and whose expression levels are predictive of cancer progression. Surprisingly, we find that several commonly studied RNA modification enzymes such as METTL3 or FTO are not significantly upregulated in most cancer types, whereas several less-characterized RMPs, such as LAGE3 and HENMT1, are dysregulated in many cancers. Conclusions: Our analyses reveal an unanticipated heterogeneity in the expression patterns of RMPs across mammalian tissues and uncover a large proportion of dysregulated RMPs in multiple cancer types. We provide novel targets for future cancer research studies targeting the human epitranscriptome, as well as foundations to understand cell type-specific behaviors that are orchestrated by RNA modifications.

The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimer's and Parkinson's disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within non-coding regions of the genome, hindering their functional validation. While some of these GWAS loci may contain cis-acting regulatory DNA elements such as enhancers, we hypothesized that many are also transcribed into non-coding RNAs that are missing from publicly available transcriptome annotations. Here, we use targeted RNA capture ('RNA CaptureSeq') in combination with nanopore long-read cDNA sequencing to transcriptionally profile 1,023 haplotype blocks across the genome containing non-coding GWAS SNPs associated with neuropsychiatric traits, using post-mortem human brain tissue from three neurologically healthy donors. We find that the majority (62%) of targeted haplotype blocks, including 13% of intergenic blocks, are transcribed into novel, multi-exonic RNAs, most of which are not yet recorded in GENCODE annotations. We validated our findings with short-read RNA-seq, providing orthogonal confirmation of novel splice junctions and enabling a quantitative assessment of the long-read assemblies. Many novel transcripts are supported by independent evidence of transcription including cap analysis of gene expression (CAGE) data and epigenetic marks, and some show signs of potential functional roles. We present these transcriptomes as a preliminary atlas of non-coding transcription in human brain that can be used to connect neurological phenotypes with gene expression.

The epitranscriptomics field has undergone an enormous expansion in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here, we show that using direct RNA sequencing, N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Specifically, we find that our algorithm, trained with m6A-modified and unmodified synthetic sequences, can predict m6A RNA modifications with ~90% accuracy. We then extend our findings to yeast data sets, finding that our method can identify m6A RNA modifications in vivo with an accuracy of 87%. Moreover, we further validate our method by showing that these ‘errors’ are typically not observed in yeast ime4-knockout strains, which lack m6A modifications. Our results open avenues to investigate the biological roles of RNA modifications in their native RNA context.

Transcriptomic studies have demonstrated that the vast majority of the genomes of mammals and other complex organisms is expressed in highly dynamic and cell-specific patterns to produce large numbers of intergenic, antisense and intronic long non-protein-coding RNAs (lncRNAs). Despite well characterized examples, their scaling with developmental complexity, and many demonstrations of their association with cellular processes, development and diseases, lncRNAs are still to be widely accepted as major players in gene regulation. This may reflect an underappreciation of the extent and precision of the epigenetic control of differentiation and development, where lncRNAs appear to have a central role, likely as organizational and guide molecules: most lncRNAs are nuclear-localized and chromatin-associated, with some involved in the formation of specialized subcellular domains. I suggest that a reassessment of the conceptual framework of genetic information and gene expression in the 4-dimensional ontogeny of spatially organized multicellular organisms is required. Together with this and further studies on their biology, the key challenges now are to determine the structure-function relationships of lncRNAs, which may be aided by emerging evidence of their modular structure, the role of RNA editing and modification in enabling epigenetic plasticity, and the role of RNA signaling in transgenerational inheritance of experience.

The amount of regulatory RNA encoded in the genome and the extent of RNA editing by the post-transcriptional deamination of adenosine to inosine (A-I) have increased with developmental complexity and may be an important factor in the cognitive evolution of animals. The newest member of the A-I editing family of ADAR proteins, the vertebrate-specific ADAR3, is highly expressed in the brain, but its functional significance is unknown. In vitro studies have suggested that ADAR3 acts as a negative regulator of A-I RNA editing but the scope and underlying mechanisms are also unknown. Meta-analysis of published data indicates that mouse Adar3 expression is highest in the hippocampus, thalamus, amygdala, and olfactory region. Consistent with this, we show that mice lacking exon 3 of Adar3 (which encodes two double stranded RNA binding domains) have increased levels of anxiety and deficits in hippocampus-dependent short- and long-term memory formation. RNA sequencing revealed a dysregulation of genes involved in synaptic function in the hippocampi of Adar3-deficient mice. We also show that ADAR3 transiently translocates from the cytoplasm to the nucleus upon KCl-mediated activation in SH-SY5Y cells. These results indicate that ADAR3 contributes to cognitive processes in mammals.

The human transcriptome is so large, diverse, and dynamic that, even after a decade of investigation by RNA sequencing (RNA-seq), we have yet to resolve its true dimensions. RNA-seq suffers from an expression-dependent bias that impedes characterization of low-abundance transcripts. We performed targeted single-molecule and short-read RNA-seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome, identifying a fundamental distinction between protein-coding and noncoding gene content: almost every noncoding exon undergoes alternative splicing, producing a seemingly limitless variety of isoforms. Analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse, yet human splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a deeply conserved splicing code. We propose that noncoding exons are functionally modular, with alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution.

In many vertebrates, sex of offspring is determined by external environmental cues rather than by sex chromosomes. In reptiles, for instance, temperature-dependent sex determination (TSD) is common. Despite decades of work, the mechanism by which temperature is converted into a sex-determining signal remains mysterious. This is partly because it is difficult to distinguish the primary molecular events of TSD from the confounding downstream signatures of sexual differentiation. We use the Australian central bearded dragon, in which chromosomal sex determination is overridden at high temperatures to produce sex-reversed female offspring, as a unique model to identify TSD-specific features of the transcriptome. We show that an intron is retained in mature transcripts from each of two Jumonji family genes, JARID2 and JMJD3, in female dragons that have been sex-reversed by temperature but not in normal chromosomal females or males. JARID2 is a component of the master chromatin modifier Polycomb Repressive Complex 2, and the mammalian sex-determining factor SRY is directly regulated by an independent but closely related Jumonji family member. We propose that the perturbation of JARID2/JMJD3 function by intron retention alters the epigenetic landscape to override chromosomal sex-determining cues, triggering sex reversal at extreme temperatures. Sex reversal may then facilitate a transition from genetic sex determination to TSD, with JARID2/JMJD3 intron retention preserved as the decisive regulatory signal. Significantly, we also observe sex-associated differential retention of the equivalent introns in JARID2/JMJD3 transcripts expressed in embryonic gonads from TSD alligators and turtles, indicative of a reptile-wide mechanism controlling TSD.

© 2017 The Author(s). Background: New approaches are required to address the needs of complex undiagnosed diseases patients. These approaches include clinical genomic diagnostic pipelines, utilizing intra- and multi-disciplinary platforms, as well as specialty-specific genomic clinics. Both are advancing diagnostic rates. However, complementary cross-disciplinary approaches are also critical to address those patients with multisystem disorders who traverse the bounds of multiple specialties and remain undiagnosed despite existing intra-specialty and genomic-focused approaches. The diagnostic possibilities of undiagnosed diseases include genetic and non-genetic conditions. The focus on genetic diseases addresses some of these disorders, however a cross-disciplinary approach is needed that also simultaneously addresses other disorder types. Herein, we describe the initiation and summary outcomes of a public health system approach for complex undiagnosed patients- the Undiagnosed Diseases Program-Western Australia (UDP-WA). Results: Briefly the UDP-WA is: i) one of a complementary suite of approaches that is being delivered within health service, and with community engagement, to address the needs of those with severe undiagnosed diseases; ii) delivered within a public health system to support equitable access to health care, including for those from remote and regional areas; iii) providing diagnoses and improved patient care; iv) delivering a platform for in-service and real time genomic and phenomic education for clinicians that traverses a diverse range of specialties; v) retaining and recapturing clinical expertise; vi) supporting the education of junior and more senior medical staff; vii) designed to integrate with clinical translational research; and viii) is supporting greater connectedness for patients, families and medical staff. Conclusion: The UDP-WA has been initiated in the public health system to complement existing clinical genomic approaches; it has be

© 2017 Ashraf et al. RNA modifications have been historically considered as fine-tuning chemo-structural features of infrastructural RNAs, such as rRNAs, tRNAs, and snoRNAs. This view has changed dramatically in recent years, to a large extent as a result of systematic efforts to map and quantify various RNA modifications in a transcriptome-wide manner, revealing that RNA modifications are reversible, dynamically regulated, far more widespread than originally thought, and involved in major biological processes, including cell differentiation, sex determination, and stress responses. Here we summarize the state of knowledge and provide a catalog of RNA modifications and their links to neurological disorders, cancers, and other diseases. With the advent of direct RNA-sequencing technologies,weexpect that this catalog will help prioritize thoseRNAmodifications for transcriptome-wide maps.

© 2017 Macmillan Publishers Limited, part of Springer Nature. RNA modifications can alter RNA structure-function relationships and various cellular processes. However, the genomic distribution and biological roles of most RNA modifications remain uncharacterized. Here, we propose using phage display antibody technology and direct sequencing through nanopores to facilitate systematic interrogation of the distribution, location and dynamics of RNA modifications.

Despite their abundance, the molecular functions of long non-coding RNAs in mammalian nervous systems remain poorly understood. Here we show that the long non-coding RNA, NEAT1, directly modulates neuronal excitability and is associated with pathological seizure states. Specifically, NEAT1 is dynamically regulated by neuronal activity in vitro and in vivo, binds epilepsy-associated potassium channel-interacting proteins including KCNAB2 and KCNIP1, and induces a neuronal hyper-potentiation phenotype in iPSC-derived human cortical neurons following antisense oligonucleotide knockdown. Next generation sequencing reveals a strong association of NEAT1 with increased ion channel gene expression upon activation of iPSC-derived neurons following NEAT1 knockdown. Furthermore, we show that while NEAT1 is acutely down-regulated in response to neuronal activity, repeated stimulation results in NEAT1 becoming chronically unresponsive in independent in vivo rat model systems relevant to temporal lobe epilepsy. We extended previous studies showing increased NEAT1 expression in resected cortical tissue from high spiking regions of patients suffering from intractable seizures. Our results indicate a role for NEAT1 in modulating human neuronal activity and suggest a novel mechanistic link between an activity-dependent long non-coding RNA and epilepsy.

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.

Targeted RNA sequencing (CaptureSeq) uses oligonucleotide probes to capture RNAs for sequencing, providing enriched read coverage, accurate measurement of gene expression, and quantitative expression data. We applied CaptureSeq to refine transcript annotations in the current murine GRCm38 assembly. More than 23,000 regions corresponding to putative or annotated long noncoding RNAs (lncRNAs) and 154,281 known splicing junction sites were selected for targeted sequencing across five mouse tissues and three brain subregions. The results illustrate that the mouse transcriptome is considerably more complex than previously thought. We assemble more complete transcript isoforms than GENCODE, expand transcript boundaries, and connect interspersed islands of mapped reads. We describe a novel filtering pipeline that identifies previously unannotated but high-quality transcript isoforms. In this set, 911 GENCODE neighboring genes are condensed into 400 expanded gene models. Additionally, 594 GENCODE lncRNAs acquire an open reading frame (ORF) when their structure is extended with CaptureSeq. Finally, we validate our observations using current FANTOM and Mouse ENCODE resources.

During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations.

Background Patients with neuroblastoma due to the amplification of a 130-kb genomic DNA region containing the MYCN oncogene have poor prognoses. Methods Bioinformatics data were used to discover a novel long noncoding RNA, lncUSMycN, at the 130-kb amplicon. RNA-protein pull-down assays were used to identify proteins bound to lncUSMycN RNA. Kaplan-Meier survival analysis, multivariable Cox regression, and two-sided log-rank test were used to examine the prognostic value of lncUSMycN and NonO expression in three cohorts of neuroblastoma patients (n = 47, 88, and 476, respectively). Neuroblastoma-bearing mice were treated with antisense oligonucleotides targeting lncUSMycN (n = 12) or mismatch sequence (n = 13), and results were analyzed by multiple comparison two-way analysis of variance. All statistical tests were two-sided. Results Bioinformatics data predicted lncUSMycN gene and RNA, and reverse-transcription polymerase chain reaction confirmed its three exons and two introns. The lncUSMycN gene was coamplified with MYCN in 88 of 341 human neuroblastoma tissues. lncUSMycN RNA bound to the RNA-binding protein NonO, leading to N-Myc RNA upregulation and neuroblastoma cell proliferation. High levels of lncUSMycN and NonO expression in human neuroblastoma tissues independently predicted poor patient prognoses (lncUSMycN: hazard ratio [HR] = 1.87, 95% confidence interval [CI] = 1.06 to 3.28, P =. 03; NonO: HR = 2.48, 95% CI = 1.34 to 4.57, P =. 004). Treatment with antisense oligonucleotides targeting lncUSMycN in neuroblastoma-bearing mice statistically significantly hindered tumor progression (P <. 001). Conclusions Our data demonstrate the important roles of lncUSMycN and NonO in regulating N-Myc expression and neuroblastoma oncogenesis and provide the first evidence that amplification of long noncoding RNA genes can contribute to tumorigenesis. © 2014 The Author 2014. Published by Oxford University Press. All rights reserved.

Despite recent controversies, the evidence that the majority of the human genome is transcribed into RNA remains strong.

Large numbers of long RNAs with little or no protein-coding potential [long noncoding RNAs (lncRNAs)] are being identified in eukaryotes. In parallel, increasing data describing the expression profiles, molecular features and functions of individual lncRNAs in a variety of systems are accumulating. To enable the systematic compilation and updating of this information, we have developed a database (lncRNAdb) containing a comprehensive list of lncRNAs that have been shown to have, or to be associated with, biological functions in eukaryotes, as well as messenger RNAs that have regulatory roles. Each entry contains referenced information about the RNA, including sequences, structural information, genomic context, expression, subcellular localization, conservation, functional evidence and other relevant information. lncRNAdb can be searched by querying published RNA names and aliases, sequences, species and associated protein-coding genes, as well as terms contained in the annotations, such as the tissues in which the transcripts are expressed and associated diseases. In addition, lncRNAdb is linked to the UCSC Genome Browser for visualization and Noncoding RNA Expression Database (NRED) for expression information from a variety of sources. lncRNAdb provides a platform for the ongoing collation of the literature pertaining to lncRNAs and their association with other genomic elements. lncRNAdb can be accessed at: http://www.lncrnadb.org/.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

The 3' untranslated regions (3'UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3'UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3'UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3'UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3'UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3'UTRs, as well as caution in the use of 3'UTR sequence probes to analyze gene expression.

BACKGROUND: The increasing interest in small non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) and recent advances in sequencing technology have yielded large numbers of short (18-32 nt) RNA sequences from different organisms, some of which are derived from small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs). We observed that these short ncRNAs frequently cover the entire length of annotated snoRNAs or tRNAs, which suggests that other loci specifying similar ncRNAs can be identified by clusters of short RNA sequences. RESULTS: We combined publicly available datasets of tens of millions of short RNA sequence tags from Drosophila melanogaster, and mapped them to the Drosophila genome. Approximately 6 million perfectly mapping sequence tags were then assembled into 521,302 tag-contigs (TCs) based on tag overlap. Most transposon-derived sequences, exons and annotated miRNAs, tRNAs and snoRNAs are detected by TCs, which show distinct patterns of length and tag-depth for different categories. The typical length and tag-depth of snoRNA-derived TCs was used to predict 7 previously unrecognized box H/ACA and 26 box C/D snoRNA candidates. We also identified one snRNA candidate and 86 loci with a high number of tags that are yet to be annotated, 7 of which have a particular 18mer motif and are located in introns of genes involved in development. A subset of new snoRNA candidates and putative ncRNA candidates was verified by Northern blot. CONCLUSIONS: In this study, we have introduced a new approach to identify new members of known classes of ncRNAs based on the features of TCs corresponding to known ncRNAs. A large number of the identified TCs are yet to be examined experimentally suggesting that many more novel ncRNAs remain to be discovered.

In mammals, thousands of long non-protein-coding RNAs (ncRNAs) (>200 nt) have recently been described. However, the biological significance and function of the vast majority of these transcripts remain unclear. We have constructed a public repository, the Noncoding RNA Expression Database (NRED), which provides gene expression information for thousands of long ncRNAs in human and mouse. The database contains both microarray and in situ hybridization data, much of which is described here for the first time. NRED also supplies a rich tapestry of ancillary information for featured ncRNAs, including evolutionary conservation, secondary structure evidence, genomic context links and antisense relationships. The database is available at http://jsm-research.imb.uq.edu.au/NRED, and the web interface enables both advanced searches and data downloads. Taken together, NRED should significantly advance the study and understanding of long ncRNAs, and provides a timely and valuable resource to the scientific community.

The assumption that RNA can be readily classified into either protein-coding or non-protein-coding categories has pervaded biology for close to 50 years. Until recently, discrimination between these two categories was relatively straightforward: most transcripts were clearly identifiable as protein-coding messenger RNAs (mRNAs), and readily distinguished from the small number of well-characterized non-protein-coding RNAs (ncRNAs), such as transfer, ribosomal, and spliceosomal RNAs. Recent genome-wide studies have revealed the existence of thousands of noncoding transcripts, whose function and significance are unclear. The discovery of this hidden transcriptome and the implicit challenge it presents to our understanding of the expression and regulation of genetic information has made the need to distinguish between mRNAs and ncRNAs both more pressing and more complicated. In this Review, we consider the diverse strategies employed to discriminate between protein-coding and noncoding transcripts and the fundamental difficulties that are inherent in what may superficially appear to be a simple problem. Misannotations can also run in both directions: some ncRNAs may actually encode peptides, and some of those currently thought to do so may not. Moreover, recent studies have shown that some RNAs can function both as mRNAs and intrinsically as functional ncRNAs, which may be a relatively widespread phenomenon. We conclude that it is difficult to annotate an RNA unequivocally as protein-coding or noncoding, with overlapping protein-coding and noncoding transcripts further confounding this distinction. In addition, the finding that some transcripts can function both intrinsically at the RNA level and to encode proteins suggests a false dichotomy between mRNAs and ncRNAs. Therefore, the functionality of any transcript at the RNA level should not be discounted.

RNAdb is a comprehensive database of mammalian non-protein-coding RNAs (ncRNAs). There is increasing recognition that ncRNAs play important regulatory roles in multicellular organisms, and there is an expanding rate of discovery of novel ncRNAs as well as an increasing allocation of function. In this update to RNAdb, we provide nucleotide sequences and annotations for tens of thousands of non-housekeeping ncRNAs, including a wide range of mammalian microRNAs, small nucleolar RNAs and larger mRNA-like ncRNAs. Some of these have documented functions and/or expression patterns, but the majority remain of unclear significance, and include PIWI-interacting RNAs, ncRNAs identified from the latest rounds of large-scale cDNA sequencing projects, putative antisense transcripts, as well as ncRNAs predicted on the basis of structural features and alignments. Improvements to the database comprise not only new and updated ncRNA datasets, but also provision of microarray-based expression data and closer interface with more specialized ncRNA resources such as miRBase and snoRNA-LBME-db. To access RNAdb, visit http://research.imb.uq.edu.au/RNAdb.

Non-protein-coding RNAs (ncRNAs) are increasingly being recognized as having important regulatory roles. Although much recent attention has focused on tiny 22- to 25-nucleotide microRNAs, several functional ncRNAs are orders of magnitude larger in size. Examples of such macro ncRNAs include Xist and Air, which in mouse are 18 and 108 kilobases (Kb), respectively. We surveyed the 102,801 FANTOM3 mouse cDNA clones and found that Air and Xist were present not as single, full-length transcripts but as a cluster of multiple, shorter cDNAs, which were unspliced, had little coding potential, and were most likely primed from internal adenine-rich regions within longer parental transcripts. We therefore conducted a genome-wide search for regional clusters of such cDNAs to find novel macro ncRNA candidates. Sixty-six regions were identified, each of which mapped outside known protein-coding loci and which had a mean length of 92 Kb. We detected several known long ncRNAs within these regions, supporting the basic rationale of our approach. In silico analysis showed that many regions had evidence of imprinting and/or antisense transcription. These regions were significantly associated with microRNAs and transcripts from the central nervous system. We selected eight novel regions for experimental validation by northern blot and RT-PCR and found that the majority represent previously unrecognized noncoding transcripts that are at least 10 Kb in size and predominantly localized in the nucleus. Taken together, the data not only identify multiple new ncRNAs but also suggest the existence of many more macro ncRNAs like Xist and Air.

In recent years, there have been increasing numbers of transcripts identified that do not encode proteins, many of which are developmentally regulated and appear to have regulatory functions. Here, we describe the construction of a comprehensive mammalian noncoding RNA database (RNAdb) which contains over 800 unique experimentally studied non-coding RNAs (ncRNAs), including many associated with diseases and/or developmental processes. The database is available at http://research.imb.uq.edu.au/RNAdb and is searchable by many criteria. It includes microRNAs and snoRNAs, but not infrastructural RNAs, such as rRNAs and tRNAs, which are catalogued elsewhere. The database also includes over 1100 putative antisense ncRNAs and almost 20,000 putative ncRNAs identified in high-quality murine and human cDNA libraries, with more to be added in the near future. Many of these RNAs are large, and many are spliced, some alternatively. The database will be useful as a foundation for the emerging field of RNomics and the characterization of the roles of ncRNAs in mammalian gene expression and regulation.

Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.

Twitching motility is a form of surface translocation mediated by the extension, tethering, and retraction of type IV pili. Three independent Tn5-B21 mutations of Pseudomonas aeruginosa with reduced twitching motility were identified in a new locus which encodes a predicted protein of unknown function annotated PA4959 in the P. aeruginosa genome sequence. Complementation of these mutants with the wild-type PA4959 gene, which we designated fimX, restored normal twitching motility. fimX mutants were found to express normal levels of pilin and remained sensitive to pilus-specific bacteriophages, but they exhibited very low levels of surface pili, suggesting that normal pilus function was impaired. The fimX gene product has a molecular weight of 76,000 and contains four predicted domains that are commonly found in signal transduction proteins: a putative response regulator (CheY-like) domain, a PAS-PAC domain (commonly involved in environmental sensing), and DUF1 (or GGDEF) and DUF2 (or EAL) domains, which are thought to be involved in cyclic di-GMP metabolism. Red fluorescent protein fusion experiments showed that FimX is located at one pole of the cell via sequences adjacent to its CheY-like domain. Twitching motility infimX mutants was found to respond relatively normally to a range of environmental factors but could not be stimulated by tryptone and mucin. These data suggest that fimX is involved in the regulation of twitching motility in response to environmental cues.

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.

Using the complete genome sequence of Pseudomonas aeruginosa PAO1, sequenced by the Pseudomonas Genome Project (ftp://ftp.pseudomonas.com/data/pacontigs.121599), a genome database (http://pseudomonas.bit.uq.edu.au/) has been developed containing information on more than 95% of all ORFs in Pseudomonas aeruginosa. The database is searchable by a variety of means, including gene name, position, keyword, sequence similarity and Pfam domain. Automated and manual annotation, nucleotide and peptide sequences, Pfam and SMART domains (where available), Medline and GenBank links and a scrollable, graphical representation of the surrounding genomic landscape are available for each ORF. Using the database has revealed, among other things, that P. aeruginosa contains four chemotaxis systems, two novel general secretion pathways, at least three loci encoding F17-like thin fimbriae, six novel filamentous haemagglutinin-like genes, a number of unusual composite genetic loci related to vgr/RHs elements in Escherichia coli, a number of fix-like genes encoding a micro-oxic respiration system, novel biosynthetic pathways and 38 genes containing domains of unknown function (DUF1/DUF2). It is anticipated that this database will be a useful bioinformatic tool for the Pseudomonas community that will continue to evolve.

Twitching motility is a form of solid surface translocation which occurs in a wide range of bacteria and which is dependent on the presence of functional type IV fimbriae or pili. A detailed examination of twitching motility in Pseudomonas aeruginosa under optimal conditions in vitro was carried out. Under these conditions (at the smooth surface formed between semi-solid growth media and plastic or glass surfaces) twitching motility is extremely rapid, leading to an overall radial rate of colony expansion of 0.6 mm h-1 or greater. The zones of colony expansion due to twitching motility are very thin and are best visualized by staining. These zones exhibit concentric rings in which there is a high density of microcolonies, which may reflect periods of expansion and consolidation/cell division. Video microscopic analysis showed that twitching motility involves the initial formation of large projections or rafts of aggregated cells which move away from the colony edge. Behind the rafts, individual cells move rapidly up and down trails which thin and branch out, ultimately forming a fine lattice-like network of cells. The bacteria in the lattice network then appear to settle and divide to fill out the colonized space. Our observations redefine twitching motility as a rapid, highly organized mechanism of bacterial translocation by which P. aeruginosa can disperse itself over large areas to colonize new territories. It is also now clear, both morphologically and genetically, that twitching motility and social gliding motility, such as occurs in Myxococcus xanthus, are essentially the same process.

Type IV pili of the opportunistic pathogen Pseudomonas aeruginosa mediate twitching motility and act as receptors for bacteriophage infection. They are also important bacterial adhesins, and nonpiliated mutants of P. aeruginosa have been shown to cause less epithelial cell damage in vitro and have decreased virulence in animal models. This finding raises the question as to whether the reduction in cytotoxicity and virulence of nonpiliated P. aeruginosa mutants are primarily due to defects in cell adhesion or loss of twitching motility, or both. This work describes the role of PilT and PilU, putative nucleotide-binding proteins involved in pili function, in mediating epithelial cell injury in vitro and virulence in vivo. Mutants of pilT and pilU retain surface pili but have lost twitching motility. In three different epithelial cell lines, pilT or pilU mutants of the strain PAK caused less cytotoxicity than the wild-type strain but more than isogenic, nonpiliated pilA or rpoN mutants. The pilT and pilU mutants also showed reduced association with these same epithelial cell lines compared both to the wild type, and surprisingly, to apilA mutant. In a mouse model of acute pneumonia, the pilT and pilU mutants showed decreased colonization of the liver but not of the lung relative to the parental strain, though they exhibited no change in the ability to cause mortality. These results demonstrate that pilus function mediated by PilT and PilU is required for in vitro adherence and cytotoxicity toward epithelial cells and is important in virulence in vivo.

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.

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

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 phosphoribosyltranferase. Insertional mutagenesis of hopB and hopD failed to reveal any obvious effects on cell viability, morphogenesis of M 13 phage, conjugative transfer of the F plasmid, or protein secretion. © 1994.

Type-4 fimbriae (pili) are associated with a phenomenon known as twitching motility, which appears to be involved with bacterial translocation across solid surfaces. Pseudomonas aeruginosa mutants which produce fimbriae, but which have lost the twitching motility function, display altered colony morphology and resistance to fimbrial-specific bacteriophage. We have used phenotypic complementation of such mutants to isolate a region of DNA involved in twitching motility. This region was physically mapped to a SpeI fragment around 20 min on the P. aeruginosa PAO chromosome, remote from the major fimbrial locus (around 75 min) where the structural subunit-encoding gene (fimA/pilA) and ancillary genes required for fimbrial assembly (pilB, C and D) are found. A gene, pilT, within the twitching motility region is predicted to encode a 344-amino acid protein which has strong homology to a variety of other bacterial proteins. These include the P. aeruginosa PilB protein, the ComG ORF-1 protein from the Bacillus subtilis comG operon (necessary for competence), the PulE protein from the Klebsiella oxytoca (formerly K. pneumoniae) pulC-O operon (involved in pullulanase export), and the VirB-11 protein from the virB operon (involved in virulence) which is located on the Agrobacterium tumefaciens Ti plasmid. We have also identified other sets of homologies between P. aeruginosa fimbrial assembly (Pil) proteins and B. subtilis Com and K. oxytoca Pul proteins, which suggest that these are all related members of a specialised protein export pathway which is widespread in the eubacteria. © 1991.