Biomedical subjects

J Michael Thomson

Publications and source records attributed to J Michael Thomson.

12 recordsLinked to original sources

PubMed2006

Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors.

Micro-RNAs (miRNAs) are a class of non-coding RNAs that post-transcriptionally regulate gene expression via the RNA interference pathway. In addition to roles in normal development, miRNAs have recently been implicated in a range of human diseases, including cancer. We recently demonstrated that a polycistronic cluster of miRNAs, miR-17-92, is oncogenic in a mouse model for Burkitt's lymphoma. This is due, in part, to a reduced apoptotic program. In an effort to understand the regulation of miR-17-92, we have studied the promoter structure of this miRNA cluster. The primary transcript initiates from a consensus initiator sequence downstream of a nonconsensus TATA box. The core promoter region contains two functional E2F transcription factor binding sites. Chromatin immunoprecipitation demonstrates that E2F3 is the primary E2F family member that occupies the promoter. These data place miR-17-92 in a regulatory loop between E2F3 and the miR-17 target E2F1. We propose a model whereby miR-17-92 promotes cell proliferation by shifting the E2F transcriptional balance away from the pro-apoptotic E2F1 and toward the proliferative E2F3 transcriptional network.

3T3 Cells↗

PubMed2006

Early and late molecular events of glucose-induced pexophagy in Pichia pastoris require Vac8.

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like "segmented" extensions that almost completely surrounded the adjacent peroxisomes. Vac8-GFP was found at the vacuolar membrane and concentrated at the base of the arm-like protrusions that extend from the vacuole to sequester the peroxisomes. The localization of Vac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8 resulted in decreased protein stability, impaired vacuolar association and reduced degradation of peroxisomal alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a non-functional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.

Alcohol Oxidoreductases↗

PubMed2006

Extensive post-transcriptional regulation of microRNAs and its implications for cancer.

MicroRNAs (miRNAs) are short, noncoding RNAs that post-transcriptionally regulate gene expression. While hundreds of mammalian miRNA genes have been identified, little is known about the pathways that regulate the production of active miRNA species. Here we show that a large fraction of miRNA genes are regulated post-transcriptionally. During early mouse development, many miRNA primary transcripts, including the Let-7 family, are present at high levels but are not processed by the enzyme Drosha. An analysis of gene expression in primary tumors indicates that the widespread down-regulation of miRNAs observed in cancer is due to a failure at the Drosha processing step. These data uncover a novel regulatory step in miRNA function and provide a mechanism for miRNA down-regulation in cancer.

Animals↗

PubMed2005

The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation.

Understanding the molecular mechanisms that regulate cellular proliferation and differentiation is a central theme of developmental biology. MicroRNAs (miRNAs) are a class of regulatory RNAs of approximately 22 nucleotides that post-transcriptionally regulate gene expression. Increasing evidence points to the potential role of miRNAs in various biological processes. Here we show that miRNA-1 (miR-1) and miRNA-133 (miR-133), which are clustered on the same chromosomal loci, are transcribed together in a tissue-specific manner during development. miR-1 and miR-133 have distinct roles in modulating skeletal muscle proliferation and differentiation in cultured myoblasts in vitro and in Xenopus laevis embryos in vivo. miR-1 promotes myogenesis by targeting histone deacetylase 4 (HDAC4), a transcriptional repressor of muscle gene expression. By contrast, miR-133 enhances myoblast proliferation by repressing serum response factor (SRF). Our results show that two mature miRNAs, derived from the same miRNA polycistron and transcribed together, can carry out distinct biological functions. Together, our studies suggest a molecular mechanism in which miRNAs participate in transcriptional circuits that control skeletal muscle gene expression and embryonic development.

Animals↗

PubMed2005

PpATG9 encodes a novel membrane protein that traffics to vacuolar membranes, which sequester peroxisomes during pexophagy in Pichia pastoris.

When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.

Fungal Proteins↗

PubMed2005

A microRNA polycistron as a potential human oncogene.

To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene.

Animals↗

PubMed2005

Resurrecting ancestral alcohol dehydrogenases from yeast.

Modern yeast living in fleshy fruits rapidly convert sugars into bulk ethanol through pyruvate. Pyruvate loses carbon dioxide to produce acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. As many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit. We report here the resurrection of the last common ancestor of Adh1 and Adh2, called Adh(A). The kinetic behavior of Adh(A) suggests that the ancestor was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used Adh(A) to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.

Alcohol Dehydrogenase↗

PubMed2005

MicroRNAs regulate brain morphogenesis in zebrafish.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis.

Animals↗

PubMed2004

A custom microarray platform for analysis of microRNA gene expression.

MicroRNAs are short, noncoding RNA transcripts that post-transcriptionally regulate gene expression. Several hundred microRNA genes have been identified in Caenorhabditis elegans, Drosophila, plants and mammals. MicroRNAs have been linked to developmental processes in C. elegans, plants and humans and to cell growth and apoptosis in Drosophila. A major impediment in the study of microRNA function is the lack of quantitative expression profiling methods. To close this technological gap, we have designed dual-channel microarrays that monitor expression levels of 124 mammalian microRNAs. Using these tools, we observed distinct patterns of expression among adult mouse tissues and embryonic stem cells. Expression profiles of staged embryos demonstrate temporal regulation of a large class of microRNAs, including members of the let-7 family. This microarray technology enables comprehensive investigation of microRNA expression, and furthers our understanding of this class of recently discovered noncoding RNAs.

Animals↗

PubMed2004

Argonaute2 is the catalytic engine of mammalian RNAi.

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

Amino Acid Sequence↗

PubMed2003

Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins.

Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55-65 degrees C. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria, the distribution of thermophily in derived bacterial lineages, the inferred G + C content of ancient ribosomal RNA, and the geological record combined with assumptions concerning molecular clocks. The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

Bacteria↗

PubMed2002

Planetary biology--paleontological, geological, and molecular histories of life.

The history of life on Earth is chronicled in the geological strata, the fossil record, and the genomes of contemporary organisms. When examined together, these records help identify metabolic and regulatory pathways, annotate protein sequences, and identify animal models to develop new drugs, among other features of scientific and biomedical interest. Together, planetary analysis of genome and proteome databases is providing an enhanced understanding of how life interacts with the biosphere and adapts to global change.

Amino Acid Sequence↗