Biomedical subjects

Tom Maniatis

Publications and source records attributed to Tom Maniatis.

15 recordsLinked to original sources

PubMed2006

Identification of long-range regulatory elements in the protocadherin-alpha gene cluster.

The clustered protocadherins (Pcdh) are encoded by three closely linked gene clusters (Pcdh-alpha, -beta, and -gamma) that span nearly 1 million base pairs of DNA. The Pcdh-alpha gene cluster encodes a family of 14 distinct cadherin-like cell surface proteins that are expressed in neurons and are present at synaptic junctions. Individual Pcdh-alpha mRNAs are assembled from one of 14 "variable" (V) exons and three "constant" exons in a process that involves both differential promoter activation and alternative pre-mRNA splicing. In individual neurons, only one (and rarely two) of the Pcdh alpha1-12 promoters is independently and randomly activated on each chromosome. Thus, in most cells, this unusual form of monoallelic expression leads to the expression of two different Pcdh-alpha 1-12 V exons, one from each chromosome. The two remaining V exons in the cluster (Pcdh-alphaC1 and alphaC2) are expressed biallelically in every neuron. The mechanisms that underlie promoter choice and monoallelic expression in the Pcdh-alpha gene cluster are not understood. Here we report the identification of two long-range cis-regulatory elements in the Pcdh-alpha gene cluster, HS5-1 and HS7. We show that HS5-1 is required for maximal levels of expression from the Pcdh alpha1-12 and alphaC1 promoters, but not the Pcdh-alphaC2 promoter. The nearly cluster-wide requirement of the HS5-1 element is consistent with the possibility that the monoallelic expression of Pcdh-alpha V exons is a consequence of competition between individual V exon promoters for the two regulatory elements.

Animals↗

PubMed2006

Systems-level analyses identify extensive coupling among gene expression machines.

Here, we develop computational methods to assess and consolidate large, diverse protein interaction data sets, with the objective of identifying proteins involved in the coupling of multicomponent complexes within the yeast gene expression pathway. From among approximately 43 000 total interactions and 2100 proteins, our methods identify known structural complexes, such as the spliceosome and SAGA, and functional modules, such as the DEAD-box helicases, within the interaction network of proteins involved in gene expression. Our process identifies and ranks instances of three distinct, biologically motivated motifs, or patterns of coupling among distinct machineries involved in different subprocesses of gene expression. Our results confirm known coupling among transcription, RNA processing, and export, and predict further coupling with translation and nonsense-mediated decay. We systematically corroborate our analysis with two independent, comprehensive experimental data sets. The methods presented here may be generalized to other biological processes and organisms to generate principled, systems-level network models that provide experimentally testable hypotheses for coupling among biological machines.

Cluster Analysis↗

PubMed2006

Parallel pathways of virus recognition.

Viruses trigger signaling pathways of innate immunity. In this issue, it is shown that the mitochondrial antiviral signaling protein is critical for intracellular detection signaling, but is dispensable for the activation of innate immunity via Toll-like receptors.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Connecting mitochondria and innate immunity.

Viral infection results in the activation of multiple signaling pathways, but how these pathways are coordinated remains a mystery. Two studies, one published in this issue of Cell (Seth et al., 2005) and the other in Molecular Cell (Xu et al., 2005), identify a new intracellular signaling protein that is required for activating type I interferon expression in response to viral infection. In addition,Seth et al. (2005) show that the function of this protein, which they call MAVS, requires that it be localized to the mitochondria. This observation establishes an unexpected link between innate immunity and an organelle with evolutionary origins in aerobic bacteria.

Animals↗

PubMed2005

The role of ubiquitination in Drosophila innate immunity.

Infection of Drosophila by Gram-negative bacteria triggers a signal transduction pathway (the IMD pathway) culminating in the expression of genes encoding antimicrobial peptides. A key component in this pathway is a Drosophila IkappaB kinase (DmIKK) complex, which stimulates the cleavage and activation of the NF-kappaB transcription factor Relish. Activation of the DmIKK complex requires the MAP3K dTAK1, but the mechanism of dTAK1 activation is not understood. In human cells, the activation of TAK1 and IKK requires the human ubiquitin-conjugating enzymes Ubc13 and UEV1a. Here we demonstrate that the Drosophila homologs of Ubc13 and UEV1a are similarly required for the activation of dTAK1 and the DmIKK complex. Surprisingly, we find that the Drosophila caspase DREDD and its partner dFADD are required for the activation of DmIKK and JNK, in addition to their role in Relish cleavage. These studies reveal an evolutionarily conserved role of ubiquitination in IKK activation, and provide new insights into the hierarchy of signaling components in the Drosophila antibacterial immunity pathway.

Animals↗

PubMed2005

Delay in synthesis of the 3' splice site promotes trans-splicing of the preceding 5' splice site.

Premessenger RNA (pre-mRNA) splicing can occur within an individual pre-mRNA (cis-splicing) or between separate pre-mRNAs (trans-splicing). Although a number of examples of mammalian trans-splicing have been reported, the molecular mechanisms involved are poorly understood. Here, we investigate the mechanisms of Sp1 pre-mRNA trans-splicing with human cells expressing modified Sp1 transgenes. We find that the presence of a long intron or the insertion of an RNA polymerase II pause site within an intron promotes trans-splicing. We also add examples of naturally occurring trans-splicing. We propose that Sp1 trans-splicing, and other examples of mammalian trans-splicing, are a consequence of low-frequency disruption of the normal mechanisms that couple transcription and splicing.

Animals↗

PubMed2005

Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers.

The 5' and 3' splice sites within an intron can, in principle, be joined to those within any other intron during pre-mRNA splicing. However, exons are joined in a strict 5' to 3' linear order in constitutively spliced pre-mRNAs. Thus, specific mechanisms must exist to prevent the random joining of exons. Here we report that insertion of exon sequences into an intron can inhibit splicing to the downstream 3' splice site and that this inhibition is independent of intron size. The exon sequences required for splicing inhibition were found to be exonic enhancer elements, and their inhibitory activity requires the binding of serine/arginine-rich splicing factors. We conclude that exonic enhancers can act as barriers to prevent exon skipping and thereby may play a key role in ensuring the correct 5' to 3' linear order of exons in spliced mRNA.

Animals↗

PubMed2004

Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-beta enhancer.

Transcriptional activation of the interferon-beta (IFN-beta) gene requires assembly of an enhanceosome containing the transcription factors ATF-2/c-Jun, IRF-3/IRF-7, NF-kappaB and HMGI(Y). These factors cooperatively bind a composite DNA site and activate expression of the IFN-beta gene. The 3.0 A crystal structure of the DNA-binding domains of ATF-2/c-Jun and two IRF-3 molecules in a complex with 31 base pairs (bp) of the PRDIV-PRDIII region of the IFN-beta enhancer shows that association of the four proteins with DNA creates a continuous surface for the recognition of 24 bp. The structure, together with in vitro binding studies and protein mutagenesis, shows that protein-protein interactions are not critical for cooperative binding. Instead, cooperativity arises mainly through nucleotide sequence-dependent structural changes in the DNA that allow formation of complementary DNA conformations. Because the binding sites overlap on the enhancer, the unit of recognition is the entire nucleotide sequence, not the individual subsites.

Activating Transcription Factor 2↗

PubMed2003

IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts.

Virus infection, double-stranded RNA, and lipopolysaccharide each induce the expression of genes encoding IFN-alpha and -beta and chemokines, such as RANTES (regulated on activation, normal T cell expressed and secreted) and IP-10 (IFN-gamma inducible protein 10). This induction requires the coordinate activation of several transcription factors, including IFN-regulatory factor 3 (IRF3). The signaling pathways leading to IRF3 activation are triggered by the binding of pathogen-specific products to Toll-like receptors and culminate in the phosphorylation of specific serine residues in the C terminus of IRF3. Recent studies of human cell lines in culture have implicated two noncanonical IkappaB kinase (IKK)-related kinases, IKK-epsilon and Traf family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK1), in the phosphorylation of IRF3. Here, we show that purified recombinant IKK-epsilon and TBK1 directly phosphorylate the critical serine residues in IRF3. We have also examined the expression of IRF3-dependent genes in mouse embryonic fibroblasts (MEFs) derived from Tbk1(-/-) mice, and we show that TBK1 is required for the activation and nuclear translocation of IRF3 in these cells. Moreover, Tbk1(-/-) MEFs show marked defects in IFN-alpha and -beta, IP-10, and RANTES gene expression after infection with either Sendai or Newcastle disease viruses or after engagement of the Toll-like receptors 3 and 4 by double-stranded RNA and lipopolysaccharide, respectively. Finally, TRIF (TIR domain-containing adapter-inducing IFN-beta), fails to activate IRF3-dependent genes in Tbk1(-/-) MEFs. We conclude that TBK1 is essential for IRF3-dependent antiviral gene expression.

Animals↗

PubMed2003

Immune activation of NF-kappaB and JNK requires Drosophila TAK1.

Stimulation of the Drosophila immune response activates NF-kappaB and JNK signaling pathways. For example, infection by Gram-negative bacteria induces the Imd signaling pathway, leading to the activation of the NF-kappaB-like transcription factor Relish and the expression of a battery of genes encoding antimicrobial peptides. Bacterial infection also activates the JNK pathway, but the role of this pathway in the immune response has not yet been established. Genetic experiments suggest that the Drosophila homolog of the mammalian MAPK kinase kinase, TAK1 (transforming growth factor beta-activated kinase 1), activates both the JNK and NF-kappaB pathways following immune stimulation. In this report, we demonstrate that Drosophila TAK1 functions as both the Drosophila IkappaB kinase-activating kinase and the JNK kinase-activating kinase. However, we found that JNK signaling is not required for antimicrobial peptide gene expression but is required for the activation of other immune inducible genes, including Punch, sulfated, and malvolio. Thus, JNK signaling appears to play an important role in the cellular immune response and the stress response.

Animals↗

PubMed2003

Caspase-mediated processing of the Drosophila NF-kappaB factor Relish.

The NF-kappaB-like transcription factor Relish plays a central role in the innate immune response of Drosophila. Unlike other NF-kappaB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IkappaB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IkappaB kinase complex and the caspase Dredd. Here we used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IkappaB-like domain, is the site of signal-dependent cleavage. We also show physical interaction between Relish and Dredd, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the Drosophila IkappaB kinase beta. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation.

Animals↗

PubMed2003

IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway.

The transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB are required for the expression of many genes involved in the innate immune response. Viral infection, or the binding of double-stranded RNA to Toll-like receptor 3, results in the coordinate activation of IRF3 and NF-kappaB. Activation of IRF3 requires signal-dependent phosphorylation, but little is known about the signaling pathway or kinases involved. Here we report that the noncanonical IkappaB kinase homologs, IkappaB kinase-epsilon (IKKepsilon) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-kappaB activation, are also essential components of the IRF3 signaling pathway. Thus, IKKepsilon and TBK1 have a pivotal role in coordinating the activation of IRF3 and NF-kappaB in the innate immune response.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Alternative pre-mRNA splicing and proteome expansion in metazoans.

The protein coding sequences of most eukaryotic messenger RNA precursors (pre-mRNAs) are interrupted by non-coding sequences called introns. Pre-mRNA splicing is the process by which introns are removed and the protein coding elements assembled into mature mRNAs. Alternative pre-mRNA splicing selectively joins different protein coding elements to form mRNAs that encode proteins with distinct functions, and is therefore an important source of protein diversity. The elaboration of this mechanism may have had a significant role in the expansion of metazoan proteomes during evolution.

Alternative Splicing↗

PubMed2002

An extensive network of coupling among gene expression machines.

Gene expression in eukaryotes requires several multi-component cellular machines. Each machine carries out a separate step in the gene expression pathway, which includes transcription, several pre-messenger RNA processing steps and the export of mature mRNA to the cytoplasm. Recent studies lead to the view that, in contrast to a simple linear assembly line, a complex and extensively coupled network has evolved to coordinate the activities of the gene expression machines. The extensive coupling is consistent with a model in which the machines are tethered to each other to form 'gene expression factories' that maximize the efficiency and specificity of each step in gene expression.

Gene Expression↗

PubMed2002

Promoter choice determines splice site selection in protocadherin alpha and gamma pre-mRNA splicing.

A family of mammalian protocadherin (Pcdh) proteins is encoded by three closely linked gene clusters (alpha, beta, and gamma). Multiple alpha and gamma Pcdh mRNAs are expressed in distinct patterns in the nervous system and are generated by alternative pre-mRNA splicing between different "variable" exons and three "constant" exons within each cluster. We show that each Pcdh variable exon is preceded by a promoter and that promoter choice determines which variable exon is included in a Pcdh mRNA. In addition, we provide evidence that alternative splicing of variable exons within a gene cluster occurs via a cis-splicing mechanism. However, virtually every variable exon can engage in trans-splicing with constant exons from another cluster, albeit at a far lower level.

Alleles↗