Autozygome sequencing expands the horizon of human knockout research and provides novel insights into human phenotypic variation.

The use of autozygosity as a mapping tool in the search for autosomal recessive disease genes is well established. We hypothesized that autozygosity not only unmasks the recessiveness of disease causing variants, but can also reveal natural knockouts of genes with less obvious phenotypic consequences. To test this hypothesis, we exome sequenced 77 well phenotyped individuals born to first cousin parents in search of genes that are biallelically inactivated. Using a very conservative estimate, we show that each of these individuals carries biallelic inactivation of 22.8 genes on average. For many of the 169 genes that appear to be biallelically inactivated, available data support involvement in modulating metabolism, immunity, perception, external appearance and other phenotypic aspects, and appear therefore to contribute to human phenotypic variation. Other genes with biallelic inactivation may contribute in yet unknown mechanisms or may be on their way to conversion into pseudogenes due to true recent dispensability. We conclude that sequencing the autozygome is an efficient way to map the contribution of genes to human phenotypic variation that goes beyond the classical definition of disease.

Identification of the tetraspanin CD82 as a new barrier to xenotransplantation.

Significant immunological obstacles are to be negotiated before xenotransplantation becomes a clinical reality. An initial rejection of transplanted vascularized xenograft is attributed to Galα1,3Galß1,4GlcNAc-R (Galα1,3-Gal)-dependent and -independent mechanisms. Hitherto, no receptor molecule has been identified that could account for Galα1,3-Gal-independent rejection. In this study, we identify the tetraspanin CD82 as a receptor molecule for the Galα1,3-Gal-independent mechanism. We demonstrate that, in contrast to human undifferentiated myeloid cell lines, differentiated cell lines are capable of recognizing xenogeneic porcine aortic endothelial cells in a calcium-dependent manner. Transcriptome-wide analysis to identify the differentially expressed transcripts in these cells revealed that the most likely candidate of the Galα1,3-Gal-independent recognition moiety is the tetraspanin CD82. Abs to CD82 inhibited the calcium response and the subsequent activation invoked by xenogeneic encounter. Our data identify CD82 on innate immune cells as a major "xenogenicity sensor" and open new avenues of intervention to making xenotransplantation a clinical reality.

Global assessment of GU-rich regulatory content and function in the human transcriptome.

Unlike AU-rich elements (AREs) that are largely present in the 3'UTRs of many unstable mammalian mRNAs, the function and abundance of GU-rich elements (GREs) are poorly understood. We performed a genome-wide analysis and found that at least 5% of human genes contain GREs in their 3'UTRs with functional over-representation in genes involved in transcription, nucleic acid metabolism, developmental processes, and neurogenesis. GREs have similar sequence clustering patterns with AREs such as overlapping GUUUG pentamers and enrichment in 3'UTRs. Functional analysis using T-cell mRNA expression microarray data confirms correlation with mRNA destabilization. Reporter assays show that compared to AREs the ability of GREs to destabilize mRNA is modest and does not increase with the increasing number of overlapping pentamers. Naturally occurring GREs within U-rich contexts were more potent in destabilizing GFP reporter mRNAs than synthetic GREs with perfectly overlapping pentamers. Overall, we find that GREs bear a resemblance to AREs in sequence patterns but they regulate a different repertoire of genes and have different dynamics of mRNA decay. A dedicated resource on all GRE-containing genes of the human, mouse and rat genomes can be found at brp.kfshrc.edu.sa/GredOrg.

AU-rich elements regulate Drosophila gene expression.

In mammals, AU-rich elements (AREs) are critical regulators of mRNA turnover. They recruit ARE-binding proteins that inhibit or stimulate rapid mRNA degradation in response to stress or developmental cues. Using a bioinformatics approach, we have identified AREs in Drosophila melanogaster 3' untranslated regions and validated their cross-species conservation in distant Drosophila genomes. We have generated a Drosophila ARE database (D-ARED) and established that about 16% of D. melanogaster genes contain the mammalian ARE signature, an AUUUA pentamer in an A/U-rich context. Using candidate ARE genes, we show that Drosophila AREs stimulate reporter mRNA decay in cultured cells and in the physiological context of the immune response in D. melanogaster. In addition, we found that the conserved ARE-binding protein Tis11 regulates temporal gene expression through ARE-mediated decay (AMD) in D. melanogaster. Our work reveals that AREs are conserved and functional cis regulators of mRNA decay in Drosophila and highlights this organism as a novel model system to unravel in vivo the contribution of AMD to various processes.

ARED Organism: expansion of ARED reveals AU-rich element cluster variations between human and mouse.

ARED Organism represents the expansion of the adenylate uridylate (AU)-rich element (ARE)-containing human mRNA database into the transcriptomes of mouse and rat. As a result, we performed quantitative assessment of ARE conservation in human, mouse and rat transcripts. We found that a significant proportion ( approximately 25%) of human genes differ in their ARE patterns from mouse and rat transcripts. ARED-Integrated, another updated and expanded version of ARED, is a compilation of ARED versions 1.0 to 3.0 and updated version 4.0 that is devoted to human mRNAs. Thus, ARED-Integrated and ARED-Organism databases, both publicly available at http://brp.kfshrc.edu.sa/ARED, offer scientists a comprehensive view of AREs in the human transcriptome and the ability to study the comparative genomics of AREs in model organisms. This ultimately will help in inferring the biological consequences of ARE variation in these key animal models as opposed to humans, particularly, in relationships to the role of RNA stability in disease.

PromoSer: improvements to the algorithm, visualization and accessibility.

PromoSer is a web service that provides an easy and efficient approach to the batch retrieval of a large number of proximal promoters. Since its introduction last year, it has undergone continued development and expansion. At the core, there have been improvements in the filtering of the raw mRNA/EST sequences upon which all predictions are built, improvements in the alignments clustering and transcription start site prediction algorithms, and improvements in the backing database for increased performance. At the user interface level, improvements include enhanced functionality and user options, better integration with other resources on the web and a new visualization tool. PromoSer now also supports queries using a SOAP-based interface and XML-based responses. The service is publicly available at http://biowulf.bu.edu/zlab/PromoSer.

SeqVISTA: a new module of integrated computational tools for studying transcriptional regulation.

Transcriptional regulation is one of the most basic regulatory mechanisms in the cell. The accumulation of multiple metazoan genome sequences and the advent of high-throughput experimental techniques have motivated the development of a large number of bioinformatics methods for the detection of regulatory motifs. The regulatory process is extremely complex and individual computational algorithms typically have very limited success in genome-scale studies. Here, we argue the importance of integrating multiple computational algorithms and present an infrastructure that integrates eight web services covering key areas of transcriptional regulation. We have adopted the client-side integration technology and built a consistent input and output environment with a versatile visualization tool named SeqVISTA. The infrastructure will allow for easy integration of gene regulation analysis software that is scattered over the Internet. It will also enable bench biologists to perform an arsenal of analysis using cutting-edge methods in a familiar environment and bioinformatics researchers to focus on developing new algorithms without the need to invest substantial effort on complex pre- or post-processors. SeqVISTA is freely available to academic users and can be launched online at http://zlab.bu.edu/SeqVISTA/web.jnlp, provided that Java Web Start has been installed. In addition, a stand-alone version of the program can be downloaded and run locally. It can be obtained at http://zlab.bu.edu/SeqVISTA.

Site2genome: locating short DNA sequences in whole genomes.

SUMMARY: Many biological papers describe short, functional DNA sites without specifying their exact positions in the genome. We have developed a Web server that automates the tedious task of locating such sites in eukaryotic genomes, thus giving access to the context of rich annotations that are increasingly available for genome sequences. AVAILABILITY: http://zlab.bu.edu/site2genome/

PromoSer: A large-scale mammalian promoter and transcription start site identification service.

Proximal promoters have a major impact on transcriptional regulation. Studies of the sequence-based nature of this regulation usually require collection of proximal promoter sequences for large sets of co-regulated genes. We report a newly implemented web service that facilitates extraction of user specified regions around the transcription start site of all annotated human, mouse or rat genes. The transcription start sites have been identified computationally by considering alignments of a large number of partial and full-length mRNA sequences to genomic DNA, with provision for alternative promoters. The service is publicly available at http://biowulf.bu.edu/zlab/PromoSer/.