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Biomedical subjects

Ben Lehner

Publications and source records attributed to Ben Lehner.

13 recordsLinked to original sources
PubMed2006

Combinatorial RNA interference in Caenorhabditis elegans reveals that redundancy between gene duplicates can be maintained for more than 80 million years of evolution.

BACKGROUND: Systematic analyses of loss-of-function phenotypes have been carried out for most genes in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster. Although such studies vastly expand our knowledge of single gene function, they do not address redundancy in genetic networks. Developing tools for the systematic mapping of genetic interactions is thus a key step in exploring the relationship between genotype and phenotype. RESULTS: We established conditions for RNA interference (RNAi) in C. elegans to target multiple genes simultaneously in a high-throughput setting. Using this approach, we can detect the great majority of previously known synthetic genetic interactions. We used this assay to examine the redundancy of duplicated genes in the genome of C. elegans that correspond to single orthologs in S. cerevisiae or D. melanogaster and identified 16 pairs of duplicated genes that have redundant functions. Remarkably, 14 of these redundant gene pairs were duplicated before the divergence of C. elegans and C. briggsae 80-110 million years ago, suggesting that there has been selective pressure to maintain the overlap in function between some gene duplicates. CONCLUSION: We established a high throughput method for examining genetic interactions using combinatorial RNAi in C. elegans. Using this technique, we demonstrated that many duplicated genes can retain redundant functions for more than 80 million years of evolution. This provides strong support for evolutionary models that predict that genetic redundancy between duplicated genes can be actively maintained by natural selection and is not just a transient side effect of recent gene duplication events.

Animals↗
PubMed2006

Systematic mapping of genetic interactions in Caenorhabditis elegans identifies common modifiers of diverse signaling pathways.

Most heritable traits, including disease susceptibility, are affected by interactions between multiple genes. However, we understand little about how genes interact because very few possible genetic interactions have been explored experimentally. We have used RNA interference in Caenorhabditis elegans to systematically test approximately 65,000 pairs of genes for their ability to interact genetically. We identify approximately 350 genetic interactions between genes functioning in signaling pathways that are mutated in human diseases, including components of the EGF/Ras, Notch and Wnt pathways. Most notably, we identify a class of highly connected 'hub' genes: inactivation of these genes can enhance the phenotypic consequences of mutation of many different genes. These hub genes all encode chromatin regulators, and their activity as genetic hubs seems to be conserved across animals. We propose that these genes function as general buffers of genetic variation and that these hub genes may act as modifier genes in multiple, mechanistically unrelated genetic diseases in humans.

Animals↗
PubMed2006

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference.

BACKGROUND: Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms. RESULTS: We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain. CONCLUSION: A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.

Animals↗
PubMed2004

A first-draft human protein-interaction map.

BACKGROUND: Protein-interaction maps are powerful tools for suggesting the cellular functions of genes. Although large-scale protein-interaction maps have been generated for several invertebrate species, projects of a similar scale have not yet been described for any mammal. Because many physical interactions are conserved between species, it should be possible to infer information about human protein interactions (and hence protein function) using model organism protein-interaction datasets. RESULTS: Here we describe a network of over 70,000 predicted physical interactions between around 6,200 human proteins generated using the data from lower eukaryotic protein-interaction maps. The physiological relevance of this network is supported by its ability to preferentially connect human proteins that share the same functional annotations, and we show how the network can be used to successfully predict the functions of human proteins. We find that combining interaction datasets from a single organism (but generated using independent assays) and combining interaction datasets from two organisms (but generated using the same assay) are both very effective ways of further improving the accuracy of protein-interaction maps. CONCLUSIONS: The complete network predicts interactions for a third of human genes, including 448 human disease genes and 1,482 genes of unknown function, and so provides a rich framework for biomedical research.

Databases, Protein↗
PubMed2004

In search of antisense.

In recent years, natural antisense transcripts (NATs) have been implicated in many aspects of eukaryotic gene expression including genomic imprinting, RNA interference, translational regulation, alternative splicing, X-inactivation and RNA editing. Moreover, there is growing evidence to suggest that antisense transcription might have a key role in a range of human diseases. Consequently, there have been several recent attempts to identify novel NATs. To date, approximately 2500 mammalian NATs have been found, indicating that antisense transcription might be a common mechanism of regulating gene expression in human cells. There are increasingly diverse ways in which antisense transcription can regulate gene expression and evidence for the involvement of NATs in human disease is emerging. A range of bioinformatic resources could be used to assist future antisense research.

Alternative Splicing↗
PubMed2004

Protein domains enriched in mammalian tissue-specific or widely expressed genes.

In multicellular organisms some genes are expressed in essentially all tissues, whereas others are expressed predominantly in only one or a few tissues. In this study, we investigate the relationship between the tissue-specificity of gene expression and the type of protein encoded. We find that many protein domains are found to be enriched in either tissue-specific or widely expressed genes. Domains enriched in tissue-specific genes tend to be metazoan-specific; these same domains are also enriched in genes that are not essential for cell viability. These findings identify families of proteins that are probably used in the development or terminal differentiation of many different tissue types.

Animals↗
PubMed2004

Analysis of a high-throughput yeast two-hybrid system and its use to predict the function of intracellular proteins encoded within the human MHC class III region.

High-throughput (HTP) protein-interaction assays, such as the yeast two-hybrid (Y2H) system, are enormously useful in predicting the functions of novel gene-products. HTP-Y2H screens typically do not include all of the reconfirmation and specificity tests used in small-scale studies, but the effects of omitting these steps have not been assessed. We performed HTP-Y2H screens that included all standard controls, using the predicted intracellular proteins expressed from the human MHC class III region, a region of the genome associated with many autoimmune diseases. The 91 novel interactions identified provide insight into the potential functions of many MHC genes, including C6orf47, LSM2, NELF-E (RDBP), DOM3Z, STK19, PBX2, RNF5, UAP56 (BAT1), ATP6G2, LST1/f, BAT2, Scythe (BAT3), CSNK2B, BAT5, and CLIC1. Surprisingly, our results predict that 1/3 of the proteins may have a role in mRNA processing, which suggests clustering of functionally related genes within the human genome. Most importantly, our analysis shows that omitting standard controls in HTP-Y2H screens could significantly compromise data quality.

Genes, Reporter↗
PubMed2004

Technique review: how to use RNA interference.

RNA interference (RNAi) has been rapidly adopted as a general method for inhibiting gene expression in most laboratory organisms. This paper discusses how libraries of RNAi reagents are being used to perform genome-wide reverse genetic screens in both model organisms and mammalian cells. Guidelines for designing effective small interfering RNAs and appropriate controls for mammalian RNAi experiments will also be discussed.

Animals↗
PubMed2004

A protein interaction framework for human mRNA degradation.

The degradation of mRNA is an important regulatory step in the control of gene expression. However, mammalian RNA decay pathways remain poorly characterized. To provide a framework for studying mammalian RNA decay, a two-hybrid protein interaction map was generated using 54 constructs from 38 human proteins predicted to function in mRNA decay. The results provide evidence for interactions between many different proteins required for mRNA decay. Of particular interest are interactions between the poly(A) ribonuclease and the exosome and between the Lsm complex, decapping factors, and 5'-->3' exonucleases. Moreover, multiple interactions connect 5'-->3' and 3'-->5' decay proteins to each other and to nonsense-mediated decay factors, providing the opportunity for coordination between decay pathways. The interaction network also predicts the internal organization of the exosome and Lsm complexes. Additional interactions connect mRNA decay factors to many novel proteins and to proteins required for other steps in gene expression. These results provide an experimental insight into the organization of proteins required for mRNA decay and their coupling to other cellular processes, and the physiological relevance of many of these interactions are supported by their evolutionary conservation. The interactions also provide a wealth of hypotheses to guide future research on mRNA degradation and demonstrate the power of exhaustive protein interaction mapping in aiding understanding of uncharacterized protein complexes and pathways.

Carrier Proteins↗
PubMed2003

The roles of intersubunit interactions in exosome stability.

In eukaryotes, at least 10 proteins associate in a 3'-5' exonuclease complex, the exosome, which is involved in the processing of many RNA species. A recent model for the exosome placed six RNase PH-related components in a hexameric ring core structure, with three S1 domain proteins associated with the ring surface. So far, however, this model lacks experimental support. Using a combination of RNA interference, complex affinity purification, and yeast two-hybrid approaches, we show here that the RNase PH homologues are important for maintenance of complex integrity. In contrast, the S1 domain proteins are not required for complex stability, although they are required for exosome function. Our results are partially consistent with the proposed model of the exosome, but indicate a different arrangement of the RNase PH proteins.

Amino Acid Sequence↗
PubMed2002

Antisense transcripts in the human genome.

By a systematic search of vertebrate mRNA sequences, we have identified a surprisingly large number of human antisense transcripts. These data suggest that regulation of gene expression by antisense and double-stranded RNAs could be a common phenomenon in mammalian cells.

Algorithms↗