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MicroRNAs (miRNA) are small RNA molecules of approximately 20 to 22 nucleotides that reduce expression of proteins through mRNA degradation and/or translational silencing. Each known miRNA has a large number of predicted targets. Members of the let-7/miR-98 family of miRNAs are up-regulated at the end of embryonic development. Let-7 is often down-regulated early during cancer development, suggesting that let-7-regulated oncofetal genes (LOG) may become reexpressed in cancer cells. Using comparative bioinformatics, we have identified 12 conserved LOGs that include HMGA2 and IMP-1/CRD-BP. IMP-1 has growth-promoting activities through stabilization of c-myc mRNA. We experimentally confirmed that IMP-1 is a direct let-7 target that promotes cell growth and motility of tumor cells, and we confirmed by proteomics analysis that IMP-1 and HMGA2 are major miRNA targets. Our data suggest that a substantial part of the growth inhibitory activities of let-7 comes from suppressing the expression of IMP-1. LOGs could be novel therapeutic targets and potential biomarkers for cancer treatment.

Accurate and efficient splicing of eukaryotic pre-mRNAs requires recognition by trans-acting factors of a complex array of cis-acting RNA elements. Here, we developed a generalized Bayesian network to model the coevolution of splicing cis elements in diverse eukaryotic taxa. Cross-exon but not cross-intron compensatory interactions between the 5' splice site (5'ss) and 3' splice site (3'ss) were observed in human/mouse, indicating that the exon is the primary evolutionary unit in mammals. Studied plants, fungi, and invertebrates exhibited exclusively cross-intron interactions, suggesting that intron definition drives evolution in these organisms. In mammals, 5'ss strength and the strength of several classes of exonic splicing silencers (ESSs) evolved in a correlated way, whereas specific exonic splicing enhancers (ESEs), including motifs associated with hTra2, SRp55, and SRp20, evolved in a compensatory manner relative to the 5'ss and 3'ss. Interactions between specific ESS or ESE motifs were not observed, suggesting that elements bound by different factors are not commonly interchangeable. Thus, the splicing elements defining exons coevolve in a way that preserves overall exon strength, allowing specific elements to substitute for loss or weakening of others.

Background: Exposure to metal working fluid (MWF) has been associated with outbreaks of extrinsic allergic alveolitis (EAA) in the USA, with bacterial contamination of MWF being a possible cause, but is uncommon in the UK. Twelve workers developed EAA in a car engine manufacturing plant in the UK, presenting clinically between December 2003 and May 2004. This paper reports the subsequent epidemiological investigation of the whole workforce. The study had three aims: (1) to measure the extent of the outbreak by identifying other workers who may have developed EAA or other work-related respiratory diseases; (2) to provide case detection so that those affected could be treated; and (3) to provide epidemiological data to identify the cause of the outbreak., Methods: The outbreak was investigated in a three-phase cross-sectional survey of the workforce. In phase I a respiratory screening questionnaire was completed by 808/836 workers (96.7%) in May 2004. In phase II 481 employees with at least one respiratory symptom on screening and 50 asymptomatic controls were invited for investigation at the factory in June 2004. This included a questionnaire, spirometry and clinical opinion. 454/481 (94.4%) responded and 48/50 (96%) controls. Workers were identified who needed further investigation and serial measurements of peak expiratory flow (PEF). In phase III 162 employees were seen at the Birmingham Occupational Lung Disease clinic. 198 employees returned PEF records, including 141 of the 162 who attended for clinical investigation. Case definitions for diagnoses were agreed., Results: 87 workers (10.4% of the workforce) met case definitions for occupational lung disease, comprising EAA (n = 19), occupational asthma (n = 74) and humidifier fever (n = 7). 12 workers had more than one diagnosis. The peak onset of work-related breathlessness was Spring 2003. The proportion of workers affected was higher for those using MWF from a large sump (27.3%) than for those working all over the manufacturing area (7.9%) (OR = 4.39, p<0.001). Two workers had positive specific provocation tests to the used but not the unused MWF solution., Conclusions: Extensive investigation of the outbreak of EAA detected a large number of affected workers, not only with EAA but also occupational asthma. This is the largest reported outbreak in Europe. Mist from used MWF is the likely cause. In workplaces using MWF there is a need to carry out risk assessments, to monitor and maintain fluid quality, to control mist and to carry out respiratory health surveillance.

Vertebrate mRNAs are frequently targeted for post-transcriptional repression by microRNAs (miRNAs) through mechanisms involving pairing of 3' UTR seed matches to bases at the 5' end of miRNAs. Through analysis of expression array data following miRNA or siRNA overexpression or inhibition, we found that mRNA fold change increases multiplicatively (i.e., log-additively) with seed match count and that a single 8 mer seed match mediates down-regulation comparable to two 7 mer seed matches. We identified several targeting determinants that enhance seed match-associated mRNA repression, including the presence of adenosine opposite miRNA base 1 and of adenosine or uridine opposite miRNA base 9, independent of complementarity to the siRNA/miRNA. Increased sequence conservation in the approximately 50 bases 5' and 3' of the seed match and increased AU content 3' of the seed match were each independently associated with increased mRNA down-regulation. All of these determinants are enriched in the vicinity of conserved miRNA seed matches, supporting their activity in endogenous miRNA targeting. Together, our results enable improved siRNA off-target prediction, allow integrated ranking of conserved and nonconserved miRNA targets, and show that targeting by endogenous and exogenous miRNAs/siRNAs involves similar or identical determinants.

Short RNA expression in several distinct stages of T-lymphocyte development was comprehensively profiled. The total number of microRNAs (miRNAs) expressed per cell at different stages of development varies over nearly an order of magnitude in parallel with changes in total cellular RNA content, suggesting that global miRNA levels are coregulated with the translational capacity of the cell. However, individual miRNAs were dynamically regulated during T-cell development, with at least one miRNA or miRNA family overrepresented at each developmental stage. miRNA regulation in this developmental pathway is characterized by analog rather than switch-like behavior, with temporal enrichments at distinct stages of development observed against a background of constant, basal expression of the miRNA. Enrichments of these miRNAs are temporally correlated with depletions of the transcript levels of targets containing seed matches to the specific miRNAs, and may have specific functional consequences. miR-181a, which is specifically enriched at the CD4(+)CD8(+) (DP) stage of thymocyte development, can repress the expression of Bcl-2, CD69, and the T-cell receptor, all of which are coordinately involved in positive selection.

The genes that are expressed in most or all types of neurons define generic neuronal features and provide a window into the developmental origin and function of the nervous system. Few such genes (sometimes referred to as pan-neuronal or broadly expressed neuronal genes) have been defined to date and the mechanisms controlling their regulation are not well understood. As a first step in investigating their regulation, we used a computational approach to detect sequences overrepresented in their promoter elements. We identified a ten-nucleotide cis-regulatory motif shared by many broadly expressed neuronal genes and demonstrated that it is involved in control of neuronal expression. Our results further suggest that global and cell-type-specific controls likely act in concert to establish pan-neuronal gene expression. Using the newly discovered motif and genome-level gene expression data, we identified a set of 234 candidate broadly expressed genes. The known involvement of many of these genes in neurogenesis and physiology of the nervous system supports the utility of this set for future targeted analyses.

Splicing mutations that lead to devastating genetic diseases are often located in nonconserved or weakly conserved sequences that normally do not affect splicing. Thus, the underlying reason for the splicing defect is not immediately obvious. An example of this phenomenon is observed in the neurodevelopmental disease familial dysautonomia (FD), which is caused by a single-base change in the 5' splice site (5'ss) of intron 20 in the IKBKAP gene (c.2204+6T>C). This mutation, which is in the sixth position of the intron and results in exon 20 skipping, has no phenotype in many other introns. To determine why the position 6 mutation causes aberrant splicing only in certain cases, we first used an in silico approach to identify potential sequences involved in exon 20 skipping. Computational analyses of the exon 20 5'ss itself predicted that this nine-nucleotide splicing signal, even when it contains the T>C mutation, is not sufficiently weak to explain the FD phenotype. However, the computational analysis predicted that both the upstream 3' splice site (3'ss) and exon 20 contain weak splicing signals, indicating that the FD 5'ss, together with the surrounding splicing signals, are not adequate for defining exon 20. These in silico predictions were corroborated using IKBKAP minigenes in a new rapid and simple in vitro coupled RNA polymerase (RNAP) II transcription/splicing assay. Finally, the weak splicing signals that flank the T>C mutation were validated as the underlying cause of familial dysautonomia in vivo using transient transfection assays. Together, our study demonstrates the general utility of combining in silico data with an in vitro RNAP II transcription/splicing system for rapidly identifying critical sequences that underlie the numerous splicing diseases caused by otherwise silent mutations., ((c) 2006 Wiley-Liss, Inc.)

Sequence-specific recognition of nucleic-acid motifs is critical to many cellular processes. We have developed a new and general method called Neighborhood Inference (NI) that predicts sequences with activity in regulating a biochemical process based on the local density of known sites in sequence space. Applied to the problem of RNA splicing regulation, NI was used to predict hundreds of new exonic splicing enhancer (ESE) and silencer (ESS) hexanucleotides from known human ESEs and ESSs. These predictions were supported by cross-validation analysis, by analysis of published splicing regulatory activity data, by sequence-conservation analysis, and by measurement of the splicing regulatory activity of 24 novel predicted ESEs, ESSs, and neutral sequences using an in vivo splicing reporter assay. These results demonstrate the ability of NI to accurately predict splicing regulatory activity and show that the scope of exonic splicing regulatory elements is substantially larger than previously anticipated. Analysis of orthologous exons in four mammals showed that the NI score of ESEs, a measure of function, is much more highly conserved above background than ESE primary sequence. This observation indicates a high degree of selection for ESE activity in mammalian exons, with surprisingly frequent interchangeability between ESE sequences., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5' and 3' splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5' splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.

We perform a statistical analysis of solvent accessibility and hydrophobicity profiles of a representative set of proteins. The joint probability distribution is well fitted to a multivariable Gaussian, which takes a relatively simple form when expressed in terms of the Fourier transforms of the profiles. This allows us to quantify the asymmetric manner by which these profiles influence each other. For example, the alpha-helix periodicity in sequence hydrophobicity is dictated by the solvent accessibility of structures, and not vice versa, possibly indicating the faster evolution of sequences compared to structures. The decorrelated hydrophobicity and solvent accessibility profiles show distinct behaviors at long periods, where sequence hydrophobicity fluctuates less, while solvent accessibility fluctuates more than average. The correlations between the two profiles can be interpreted as the Boltzmann weight of the solvation energy at room temperature, consistent with earlier observations., (2005 Wiley-Liss, Inc.)

RNA splicing is an essential step in gene expression, and is often variable, giving rise to multiple alternatively spliced mRNA and protein isoforms from a single gene locus. The design of effective databases to support experimental and computational investigations of alternative splicing (AS) is a significant challenge. In an effort to integrate accurate exon and splice site annotation with current knowledge about splicing regulatory elements and predicted AS events, and to link information about the splicing of orthologous genes in different species, we have developed the Hollywood system. This database was built upon genomic annotation of splicing patterns of known genes derived from spliced alignment of complementary DNAs (cDNAs) and expressed sequence tags, and links features such as splice site sequence and strength, exonic splicing enhancers and silencers, conserved and non-conserved patterns of splicing, and cDNA library information for inferred alternative exons. Hollywood was implemented as a relational database and currently contains comprehensive information for human and mouse. It is accompanied by a web query tool that allows searches for sets of exons with specific splicing characteristics or splicing regulatory element composition, or gives a graphical or sequence-level summary of splicing patterns for a specific gene. A streamlined graphical representation of gene splicing patterns is provided, and these patterns can alternatively be layered onto existing information in the UCSC Genome Browser. The database is accessible at http://hollywood.mit.edu.

Thousands of mammalian messenger RNAs are under selective pressure to maintain 7-nucleotide sites matching microRNAs (miRNAs). We found that these conserved targets are often highly expressed at developmental stages before miRNA expression and that their levels tend to fall as the miRNA that targets them begins to accumulate. Nonconserved sites, which outnumber the conserved sites 10 to 1, also mediate repression. As a consequence, genes preferentially expressed at the same time and place as a miRNA have evolved to selectively avoid sites matching the miRNA. This phenomenon of selective avoidance extends to thousands of genes and enables spatial and temporal specificities of miRNAs to be revealed by finding tissues and developmental stages in which messages with corresponding sites are expressed at lower levels.

Organisms respond to perturbations in DNA replication. We characterized the global transcriptional response to inhibition of DNA replication in Bacillus subtilis. We focused on changes that were independent of the known recA-dependent global DNA damage (SOS) response. We found that overlapping sets of genes are affected by perturbations in replication elongation or initiation and that this transcriptional response serves to inhibit cell division and maintain cell viability. Approximately 20 of the operons (>50 genes) affected have potential DnaA-binding sites and are probably regulated directly by DnaA, the highly conserved replication initiation protein and transcription factor. Many of these genes have homologues and recognizable DnaA-binding sites in other bacteria, indicating that a DnaA-mediated response, elicited by changes in DNA replication status, may be conserved.

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the approximately 1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.

Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

As an important component of the innate immune system, complement provides the initial response to prevent infections by pathogenic microorganisms. Patients with dysfunction of C5 display a propensity for severe recurrent infections. In this study, we present a patient with C5 deficiency demonstrated by immunochemical and functional analyses. Direct sequencing of all C5 exons displayed no mutation of obvious functional significance, except for an A to G transition in exon 10 predicting an exchange from lysine to arginine. This sequence alteration was present in only one allele of family members with a reduced serum C5 concentration and in both alleles of the patient with almost complete C5 deficiency, suggesting that this alteration may be producing the phenotype. Recent findings indicate that distinct nucleotide sequences, termed exonic splicing enhancers (ESEs), influence the splicing process. cDNA from all family members harboring the mutated allele showed skipping of exon 10, which resulted in a premature STOP codon, explaining the lack of C5 in the propositus. Sequence analysis of the mutated region revealed the substitution to be located within an ESE, as predicted by the RESCUE-ESE program. The altered ESE sequence is located close to the 5' splicing site and also lowers the predicted strength of the splice site itself. This apparently inconsequential sequence alteration represents a noncanonical splicing mutation altering an ESE. Our finding sheds a new light on the role of putative silent/conservative mutations in disease-associated genes.

Alternative pre-mRNA splicing affects a majority of human genes and plays important roles in development and disease. Alternative splicing (AS) events conserved since the divergence of human and mouse are likely of primary biological importance, but relatively few of such events are known. Here we describe sequence features that distinguish exons subject to evolutionarily conserved AS, which we call alternative conserved exons (ACEs), from other orthologous human/mouse exons and integrate these features into an exon classification algorithm, acescan. Genome-wide analysis of annotated orthologous human-mouse exon pairs identified approximately 2,000 predicted ACEs. Alternative splicing was verified in both human and mouse tissues by using an RT-PCR-sequencing protocol for 21 of 30 (70%) predicted ACEs tested, supporting the validity of a majority of acescan predictions. By contrast, AS was observed in mouse tissues for only 2 of 15 (13%) tested exons that had EST or cDNA evidence of AS in human but were not predicted ACEs, and AS was never observed for 11 negative control exons in human or mouse tissues. Predicted ACEs were much more likely to preserve the reading frame and less likely to disrupt protein domains than other AS events and were enriched in genes expressed in the brain and in genes involved in transcriptional regulation, RNA processing, and development. Our results also imply that the vast majority of AS events represented in the human EST database are not conserved in mouse.

Exonic splicing silencers (ESSs) are cis-regulatory elements that inhibit the use of adjacent splice sites, often contributing to alternative splicing (AS). To systematically identify ESSs, an in vivo splicing reporter system was developed to screen a library of random decanucleotides. The screen yielded 141 ESS decamers, 133 of which were unique. The silencer activity of over a dozen of these sequences was also confirmed in a heterologous exon/intron context and in a second cell type. Of the unique ESS decamers, most could be clustered into groups to yield seven putative ESS motifs, some resembling known motifs bound by hnRNPs H and A1. Potential roles of ESSs in constitutive splicing were explored using an algorithm, ExonScan, which simulates splicing based on known or putative splicing-related motifs. ExonScan and related bioinformatic analyses suggest that these ESS motifs play important roles in suppression of pseudoexons, in splice site definition, and in AS.

Little is known about the patterns of intron gain and loss or the relative contributions of these two processes to gene evolution. To investigate the dynamics of intron evolution, we analyzed orthologous genes from four filamentous fungal genomes and determined the pattern of intron conservation. We developed a probabilistic model to estimate the most likely rates of intron gain and loss giving rise to these observed conservation patterns. Our data reveal the surprising importance of intron gain. Between about 150 and 250 gains and between 150 and 350 losses were inferred in each lineage. We discuss one gene in particular (encoding 1-phosphoribosyl-5-pyrophosphate synthetase) that displays an unusually high rate of intron gain in multiple lineages. It has been recognized that introns are biased towards the 5' ends of genes in intron-poor genomes but are evenly distributed in intron-rich genomes. Current models attribute this bias to 3' intron loss through a poly-adenosine-primed reverse transcription mechanism. Contrary to standard models, we find no increased frequency of intron loss toward the 3' ends of genes. Thus, recent intron dynamics do not support a model whereby 5' intron positional bias is generated solely by 3'-biased intron loss., Competing Interests: The authors have declared that no conflicts of interest exist.

Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms. For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells. Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes. The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates. However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish. Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns. Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish. Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells.

MicroRNAs are approximately 22-nucleotide (nt) RNAs processed from foldback segments of endogenous transcripts. Some are known to play important gene regulatory roles during animal and plant development by pairing to the messages of protein-coding genes to direct the post-transcriptional repression of these messages. Previously, we developed a computational method called MiRscan, which scores features related to the foldbacks, and used this algorithm to identify new miRNA genes in the nematode Caenorhabditis elegans. In the present study, to identify sequences that might be involved in processing or transcriptional regulation of miRNAs, we aligned sequences upstream and downstream of orthologous nematode miRNA foldbacks. These alignments showed a pronounced peak in sequence conservation about 200 bp upstream of the miRNA foldback and revealed a highly significant sequence motif, with consensus CTCCGCCC, that is present upstream of almost all independently transcribed nematode miRNA genes. Scoring the pattern of upstream/downstream conservation, the occurrence of this sequence motif, and orthology of host genes for intronic miRNA candidates, yielded substantial improvements in the accuracy of MiRscan. Nine new C. elegans miRNA gene candidates were validated using a PCR-sequencing protocol. As previously seen for bacterial RNA genes, sequence features outside of the RNA secondary structure can therefore be very useful for the computational identification of eukaryotic noncoding RNA genes. The total number of confidently identified nematode miRNAs now approaches 100. The improved analysis supports our previous assertion that miRNA gene identification is nearing completion in C. elegans with apparently no more than 20 miRNA genes now remaining to be identified.

Because deleterious alleles arising from mutation are filtered by natural selection, mutations that create such alleles will be underrepresented in the set of common genetic variation existing in a population at any given time. Here, we describe an approach based on this idea called VERIFY (variant elimination reinforces functionality), which can be used to assess the extent of natural selection acting on an oligonucleotide motif or set of motifs predicted to have biological activity. As an application of this approach, we analyzed a set of 238 hexanucleotides previously predicted to have exonic splicing enhancer (ESE) activity in human exons using the relative enhancer and silencer classification by unanimous enrichment (RESCUE)-ESE method. Aligning the single nucleotide polymorphisms (SNPs) from the public human SNP database to the chimpanzee genome allowed inference of the direction of the mutations that created present-day SNPs. Analyzing the set of SNPs that overlap RESCUE-ESE hexamers, we conclude that nearly one-fifth of the mutations that disrupt predicted ESEs have been eliminated by natural selection (odds ratio = 0.82 +/- 0.05). This selection is strongest for the predicted ESEs that are located near splice sites. Our results demonstrate a novel approach for quantifying the extent of natural selection acting on candidate functional motifs and also suggest certain features of mutations/SNPs, such as proximity to the splice site and disruption or alteration of predicted ESEs, that should be useful in identifying variants that might cause a biological phenotype., Competing Interests: The authors have declared that no conflicts of interest exist.

A typical gene contains two levels of information: a sequence that encodes a particular protein and a host of other signals that are necessary for the correct expression of the transcript. While much attention has been focused on the effects of sequence variation on the amino acid sequence, variations that disrupt gene processing signals can dramatically impact gene function. A variation that disrupts an exonic splicing enhancer (ESE), for example, could cause exon skipping which would result in the exclusion of an entire exon from the mRNA transcript. RESCUE-ESE, a computational approach used in conjunction with experimental validation, previously identified 238 candidate ESE hexamers in human genes. The RESCUE-ESE method has recently been implemented in three additional species: mouse, zebrafish and pufferfish. Here we describe an online ESE analysis tool (http://genes.mit.edu/burgelab/rescue-ese/) that annotates RESCUE-ESE hexamers in vertebrate exons and can be used to predict splicing phenotypes by identifying sequence changes that disrupt or alter predicted ESEs.

We propose a framework for modeling sequence motifs based on the maximum entropy principle (MEP). We recommend approximating short sequence motif distributions with the maximum entropy distribution (MED) consistent with low-order marginal constraints estimated from available data, which may include dependencies between nonadjacent as well as adjacent positions. Many maximum entropy models (MEMs) are specified by simply changing the set of constraints. Such models can be utilized to discriminate between signals and decoys. Classification performance using different MEMs gives insight into the relative importance of dependencies between different positions. We apply our framework to large datasets of RNA splicing signals. Our best models out-perform previous probabilistic models in the discrimination of human 5' (donor) and 3' (acceptor) splice sites from decoys. Finally, we discuss mechanistically motivated ways of comparing models.

Given the availability of complete genome sequences from related organisms, sequence conservation can provide important clues for predicting gene structure. In particular, one should be able to leverage information about known genes in one species to help determine the structures of related genes in another. Such an approach is appealing in that high-quality gene prediction can be achieved for newly sequenced species, such as mouse and puffer fish, using the extensive knowledge that has been accumulated about human genes. This article reports a novel approach to predicting the exon-intron structures of mouse genes by incorporating constraints from orthologous human genes using techniques that have previously been exploited in speech and natural language processing applications. The approach uses a context-free grammar to parse a training corpus of annotated human genes. A statistical training procedure produces a weighted recursive transition network (RTN) intended to capture the general features of a mammalian gene. This RTN is expanded into a finite state transducer (FST) and composed with an FST capturing the specific features of the human orthologue. This model includes a trigram language model on the amino acid sequence as well as exon length constraints. A final stage uses the free software package ClustalW to align the top n candidates in the search space. For a set of 98 orthologous human-mouse pairs, we achieved 96% sensitivity and 97% specificity at the exon level on the mouse genes, given only knowledge gleaned from the annotated human genome.

Background: Alternative pre-mRNA splicing (AS) is widely used by higher eukaryotes to generate different protein isoforms in specific cell or tissue types. To compare AS events across human tissues, we analyzed the splicing patterns of genomically aligned expressed sequence tags (ESTs) derived from libraries of cDNAs from different tissues., Results: Controlling for differences in EST coverage among tissues, we found that the brain and testis had the highest levels of exon skipping. The most pronounced differences between tissues were seen for the frequencies of alternative 3' splice site and alternative 5' splice site usage, which were about 50 to 100% higher in the liver than in any other human tissue studied. Quantifying differences in splice junction usage, the brain, pancreas, liver and the peripheral nervous system had the most distinctive patterns of AS. Analysis of available microarray expression data showed that the liver had the most divergent pattern of expression of serine-arginine protein and heterogeneous ribonucleoprotein genes compared to the other human tissues studied, possibly contributing to the unusually high frequency of alternative splice site usage seen in liver. Sequence motifs enriched in alternative exons in genes expressed in the brain, testis and liver suggest specific splicing factors that may be important in AS regulation in these tissues., Conclusions: This study distinguishes the human brain, testis and liver as having unusually high levels of AS, highlights differences in the types of AS occurring commonly in different tissues, and identifies candidate cis-regulatory elements and trans-acting factors likely to have important roles in tissue-specific AS in human cells.

MicroRNAs (miRNAs) can play important gene regulatory roles in nematodes, insects, and plants by basepairing to mRNAs to specify posttranscriptional repression of these messages. However, the mRNAs regulated by vertebrate miRNAs are all unknown. Here we predict more than 400 regulatory target genes for the conserved vertebrate miRNAs by identifying mRNAs with conserved pairing to the 5' region of the miRNA and evaluating the number and quality of these complementary sites. Rigorous tests using shuffled miRNA controls supported a majority of these predictions, with the fraction of false positives estimated at 31% for targets identified in human, mouse, and rat and 22% for targets identified in pufferfish as well as mammals. Eleven predicted targets (out of 15 tested) were supported experimentally using a HeLa cell reporter system. The predicted regulatory targets of mammalian miRNAs were enriched for genes involved in transcriptional regulation but also encompassed an unexpectedly broad range of other functions.

Redundancy of the genetic code dictates that a given protein can be encoded by a large collection of distinct mRNA species, potentially allowing mRNAs to simultaneously optimize desirable RNA structural features in addition to their protein-coding function. To determine whether natural mRNAs exhibit biases related to local RNA secondary structure, a new randomization procedure was developed, DicodonShuffle, which randomizes mRNA sequences while preserving the same encoded protein sequence, the same codon usage, and the same dinucleotide composition as the native message. Genes from 10 of 14 eubacterial species studied and one eukaryote, the yeast Saccharomyces cerevisiae, exhibited statistically significant biases in favor of local RNA structure as measured by folding free energy. Several significant associations suggest functional roles for mRNA structure, including stronger secondary structure bias in the coding regions of intron-containing yeast genes than in intronless genes, and significantly higher folding potential in polycistronic messages than in monocistronic messages in Escherichia coli. Potential secondary structure generally increased in genes from the 5' to the 3' end of E. coli operons, and secondary structure potential was conserved in homologous Salmonella typhi operons. These results are interpreted in terms of possible roles of RNA structures in RNA processing, regulation of mRNA stability, and translational control.

MicroRNAs (miRNAs) are an abundant class of tiny RNAs thought to regulate the expression of protein-coding genes in plants and animals. In the present study, we describe a computational procedure to identify miRNA genes conserved in more than one genome. Applying this program, known as MiRscan, together with molecular identification and validation methods, we have identified most of the miRNA genes in the nematode Caenorhabditis elegans. The total number of validated miRNA genes stands at 88, with no more than 35 genes remaining to be detected or validated. These 88 miRNA genes represent 48 gene families; 46 of these families (comprising 86 of the 88 genes) are conserved in Caenorhabditis briggsae, and 22 families are conserved in humans. More than a third of the worm miRNAs, including newly identified members of the lin-4 and let-7 gene families, are differentially expressed during larval development, suggesting a role for these miRNAs in mediating larval developmental transitions. Most are present at very high steady-state levels-more than 1000 molecules per cell, with some exceeding 50,000 molecules per cell. Our census of the worm miRNAs and their expression patterns helps define this class of noncoding RNAs, lays the groundwork for functional studies, and provides the tools for more comprehensive analyses of miRNA genes in other species.

MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nt long that are processed by Dicer from precursors with a characteristic hairpin secondary structure. Guidelines are presented for the identification and annotation of new miRNAs from diverse organisms, particularly so that miRNAs can be reliably distinguished from other RNAs such as small interfering RNAs. We describe specific criteria for the experimental verification of miRNAs, and conventions for naming miRNAs and miRNA genes. Finally, an online clearinghouse for miRNA gene name assignments is provided by the Rfam database of RNA families.

We introduce a model of DNA sequence evolution which can account for biases in mutation rates that depend on the identity of the neighboring bases. An analytic solution for this class of models is developed by adopting well-known methods of nonlinear dynamics. Results are presented for the CpG-methylation-deamination process, which dominates point substitutions in vertebrates. The dinucleotide frequencies generated by the model (using empirically obtained mutation rates) match the overall pattern observed in noncoding DNA. A web-based tool has been constructed to compute single- and dinucleotide frequencies for arbitrary neighbor-dependent mutation rates. Also provided is the backward procedure to infer the mutation rates using maximum likelihood analysis given the observed single- and dinucleotide frequencies. Reasonable estimates of the mutation rates can be obtained very efficiently, using generic noncoding DNA sequences as input, after masking out long homonucleotide subsequences. Our method is much more convenient and versatile to use than the traditional method of deducing mutation rates by counting mutation events in carefully chosen sequences. More generally, our approach provides a more realistic but still tractable description of noncoding genomic DNA and may be used as a null model for various sequence analysis applications.

We predict regulatory targets for 14 Arabidopsis microRNAs (miRNAs) by identifying mRNAs with near complementarity. Complementary sites within predicted targets are conserved in rice. Of the 49 predicted targets, 34 are members of transcription factor gene families involved in developmental patterning or cell differentiation. The near-perfect complementarity between plant miRNAs and their targets suggests that many plant miRNAs act similarly to small interfering RNAs and direct mRNA cleavage. The targeting of developmental transcription factors suggests that many plant miRNAs function during cellular differentiation to clear key regulatory transcripts from daughter cell lineages.

Specific short oligonucleotide sequences that enhance pre-mRNA splicing when present in exons, termed exonic splicing enhancers (ESEs), play important roles in constitutive and alternative splicing. A computational method, RESCUE-ESE, was developed that predicts which sequences have ESE activity by statistical analysis of exon-intron and splice site composition. When large data sets of human gene sequences were used, this method identified 10 predicted ESE motifs. Representatives of all 10 motifs were found to display enhancer activity in vivo, whereas point mutants of these sequences exhibited sharply reduced activity. The motifs identified enable prediction of the splicing phenotypes of exonic mutations in human genes.

Splicing of short introns by the nuclear pre-mRNA splicing machinery is thought to proceed via an "intron definition" mechanism, in which the 5' and 3' splice sites (5'ss, 3'ss, respectively) are initially recognized and paired across the intron. Here, we describe a computational analysis of sequence features involved in recognition of short introns by using available transcript data from five eukaryotes with complete or nearly complete genomic sequences. The information content of five different transcript features was measured by using methods from information theory, and Monte Carlo simulations were used to determine the amount of information required for accurate recognition of short introns in each organism. We conclude: (i) that short introns in Drosophila melanogaster and Caenorhabditis elegans contain essentially all of the information for their recognition by the splicing machinery, and computer programs that simulate splicing specificity can predict the exact boundaries of approximately 95% of short introns in both organisms; (ii) that in yeast, the 5'ss, branch signal, and 3'ss can accurately identify intron locations but do not precisely determine the location of 3' cleavage in every intron; and (iii) that the 5'ss, branch signal, and 3'ss are not sufficient to accurately identify short introns in plant and human transcripts, but that specific subsets of candidate intronic enhancer motifs can be identified in both human and Arabidopsis that contribute dramatically to the accuracy of splicing simulators.

Variation in the estimates of the number of genes encoded by the human genome (28,000-120,000) attests to the difficulty of systematically identifying human genes. Sequencing of human chromosome 22 (Chr22) provided the first comprehensive, unbiased view of an entire human chromosome, and intensive analysis of this sequence identified 545 genes and 134 pseudogenes that had similarity or identity to known proteins and/or ESTs and which were listed in the gene annotation (http://www.sanger.ac.uk/HGP/Chr22). This analysis yielded an estimate of approximately 36,000 functional expressed genes in the human genome (and 9000 pseudogenes). However, a key uncertainty in this estimate was that hundreds of additional genes beyond those annotated in the Chr22 sequence are predicted by the gene prediction program Genscan, an unknown number of which might represent additional expressed genes. To determine what fraction of these "predicted novel genes" (PNGs) represents expressed human genes, we used a sensitive RT-PCR assay to detect predicted transcripts in 17 tissues and one cell line. Our results indicate that at least 5000-9000 additional human genes which lack similarity to known genes or proteins exist in the human genome, increasing baseline gene estimates to approximately 41,000-45,000.

With the human genome sequence approaching completion, a major challenge is to identify the locations and encoded protein sequences of all human genes. To address this problem we have developed a new gene identification algorithm, GenomeScan, which combines exon-intron and splice signal models with similarity to known protein sequences in an integrated model. Extensive testing shows that GenomeScan can accurately identify the exon-intron structures of genes in finished or draft human genome sequence with a low rate of false-positives. Application of GenomeScan to 2.7 billion bases of human genomic DNA identified at least 20,000-25,000 human genes out of an estimated 30,000-40,000 present in the genome. The results show an accurate and efficient automated approach for identifying genes in higher eukaryotic genomes and provide a first-level annotation of the draft human genome.

Because of advances in automation, human genomic sequences are being deposited in public databases at a dramatic rate. However, the process of detecting genes in these sequences is still something of an art. Here we describe the implementation and testing of a relatively straightforward computational approach, the Virtual Transcribed Sequence project, which analyzes their gene content using the gene prediction program GENSCAN (GENSCAN 1.0 1,2) in combination with similarity-based methods. This approach identifies many novel human genes not found even in EST databases., (Copyright 2000 Academic Press.)

If asthma is due to work exposures there must be a relation between these exposures and the asthma. Asthma causes airway hyperresponsiveness and obstruction; the obstruction can be measured with portable meters, which usually measure peak expiratory flow, or sometimes forced expiratory volume in 1 second (FEV1). These can be measured serially (for instance 2 hourly) over several weeks at and away from work. Once occupational asthma develops, the asthma will be induced by many non-specific triggers common to non-occupational asthma. The challenge is to identify changes in peak expiratory flow due to work among other non-occupational causes. Standard statistical tests have been found to be insensitive or non-specific, principally because of the variable period for deterioration to occur after exposure, and the sometimes prolonged time for recovery to occur, such that days away from work may initially have lower measurements than days at work. A computer assisted diagnostic aid (Oasys) has been developed to separate occupational from non-occupational causes of airflow obstruction. Oasys-2 is based on a discriminant analysis, and achieved a sensitivity of 75% and a specificity of at least 94%; therefore peak expiratory flow monitoring combined with Oasys-2 analysis is better to confirm than to exclude occupational asthma. A neural network version in development has improved on this. Both have been based on expert interpretation of peak flow measurements plotted as daily maximum, mean, and minimum, with the first reading at work taken as the first reading of the day. Oasys has been evaluated with independent criteria against measurements made in a wide range of occupational situations. Oasys is sufficiently developed to be the initial method for the confirmation, although less so for exclusion of occupational asthma.

U2-type and U12-type introns are spliced by distinct spliceosomes in eukaryotic nuclei. A classification method was devised to distinguish these two types of introns based on splice site sequence properties and was used to identify 56 different genes containing U12-type introns in available genomic sequences. U12-type introns occur with consistently low frequency in diverse eukaryotic taxa but have almost certainly been lost from C. elegans. Comparisons with available homologous sequences demonstrate subtype switching of U12 introns between termini of AT-AC and GT-AG as well as conversion of introns from U12-type to U2-type and provide evidence for a fission/fusion model in which the two splicing systems evolved in separate lineages that were fused in a eukaryotic progenitor.

Genome sequencing efforts will soon generate hundreds of millions of bases of human genomic DNA containing thousands of novel genes. In the past year, the accuracy of computational gene-finding methods has improved significantly, to the point where a reasonable approximation of the gene structures within an extended genomic region can often be predicted in advance of more detailed experimental studies.