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1.
Mol Brain ; 17(1): 18, 2024 Apr 11.
Article in English | MEDLINE | ID: mdl-38605409

ABSTRACT

One of the main burdens in the treatment of diseases is imputable to the delay between the appearance of molecular dysfunctions in the first affected disease cells and their presence in sufficient number for detection in specific tissues or organs. This delay obviously plays in favor of disease progression to an extent that makes efficient treatments difficult, as they arrive too late. The development of a novel medical strategy, termed cell-based interception and precision medicine, seeks to identify dysfunctional cells early, when tissue damages are not apparent and symptoms not yet present, and develop therapies to treat diseases early. Central to this strategy is the use of single-cell technologies that allow detection of molecular changes in cells at the time of phenotypical bifurcation from health to disease. In this article we describe a general procedure to support such an approach applied to neurodegenerative disorders. This procedure combines four components directed towards highly complementary objectives: 1) a high-performance single-cell proteomics (SCP) method (Detect), 2) the development of disease experimental cell models and predictive computational models of cell trajectories (Understand), 3) the discovery of specific targets and personalized therapies (Cure), and 4) the creation of a community of collaborating laboratories to accelerate the development of this novel medical paradigm (Collaborate). A global initiative named 37TrillionCells (37TC) was launched to advance the development of cell-based interception and precision medicine.


Subject(s)
Neurodegenerative Diseases , Humans , Neurodegenerative Diseases/therapy , Precision Medicine/methods , Delivery of Health Care , Proteomics/methods
2.
Front Mol Neurosci ; 17: 1371145, 2024.
Article in English | MEDLINE | ID: mdl-38571813

ABSTRACT

The prevailing model behind synapse development and specificity is that a multitude of adhesion molecules engage in transsynaptic interactions to induce pre- and postsynaptic assembly. How these extracellular interactions translate into intracellular signal transduction for synaptic assembly remains unclear. Here, we focus on a synapse organizing complex formed by immunoglobulin superfamily member 21 (IgSF21) and neurexin2α (Nrxn2α) that regulates GABAergic synapse development in the mouse brain. We reveal that the interaction between presynaptic Nrxn2α and postsynaptic IgSF21 is a high-affinity receptor-ligand interaction and identify a binding interface in the IgSF21-Nrxn2α complex. Despite being expressed in both dendritic and somatic regions, IgSF21 preferentially regulates dendritic GABAergic presynaptic differentiation whereas another canonical Nrxn ligand, neuroligin2 (Nlgn2), primarily regulates perisomatic presynaptic differentiation. To explore mechanisms that could underlie this compartment specificity, we targeted multiple signaling pathways pharmacologically while monitoring the synaptogenic activity of IgSF21 and Nlgn2. Interestingly, both IgSF21 and Nlgn2 require c-jun N-terminal kinase (JNK)-mediated signaling, whereas Nlgn2, but not IgSF21, additionally requires CaMKII and Src kinase activity. JNK inhibition diminished de novo presynaptic differentiation without affecting the maintenance of formed synapses. We further found that Nrxn2α knockout brains exhibit altered synaptic JNK activity in a sex-specific fashion, suggesting functional linkage between Nrxns and JNK. Thus, our study elucidates the structural and functional relationship of IgSF21 with Nrxn2α and distinct signaling pathways for IgSF21-Nrxn2α and Nlgn2-Nrxn synaptic organizing complexes in vitro. We therefore propose a revised hypothesis that Nrxns act as molecular hubs to specify synaptic properties not only through their multiple extracellular ligands but also through distinct intracellular signaling pathways of these ligands.

3.
bioRxiv ; 2023 Sep 12.
Article in English | MEDLINE | ID: mdl-37745343

ABSTRACT

TFIIH is an essential transcription initiation factor for RNA polymerase II (RNApII). This multi-subunit complex comprises two modules that are physically linked by the subunit Tfb3 (MAT1 in metazoans). The TFIIH Core Module, with two DNA-dependent ATPases and several additional subunits, promotes DNA unwinding. The TFIIH Kinase Module phosphorylates Serine 5 of the C-terminal domain (CTD) of RNApII subunit Rpb1, a modification that coordinates exchange of initiation and early elongation factors. While it is not obvious why these two disparate activities are bundled into one factor, the connection may provide temporal coordination during early initiation. Here we show that Tfb3 can be split into two parts to uncouple the TFIIH modules. The resulting cells grow slower than normal, but are viable. Chromatin immunoprecipitation of the split TFIIH shows that the Core Module, but not the Kinase, is properly recruited to promoters. Instead of the normal promoter-proximal peak, high CTD Serine 5 phosphorylation is seen throughout transcribed regions. Therefore, coupling the TFIIH modules is necessary to localize and limit CTD kinase activity to early stages of transcription. These results are consistent with the idea that the two TFIIH modules began as independent functional entities that became connected by Tfb3 during early eukaryotic evolution.

4.
Mol Brain ; 15(1): 98, 2022 11 30.
Article in English | MEDLINE | ID: mdl-36451185

ABSTRACT

The mechanism of assembly of RNA polymerase III (Pol III), the 17-subunit enzyme that synthesizes tRNAs, 5 S rRNA, and other small-nuclear (sn) RNAs in eukaryotes, is not clearly understood. The recent discovery of the HSP90 co-chaperone PAQosome (Particle for Arrangement of Quaternary structure) revealed a function for this machinery in the biogenesis of nuclear RNA polymerases. However, the connection between Pol III subunits and the PAQosome during the assembly process remains unexplored. Here, we report the development of a mass spectrometry-based assay that allows the characterization of Pol III assembly. This assay was used to dissect the stages of Pol III assembly, to start defining the function of the PAQosome in this process, to dissect the assembly defects driven by the leukodystrophy-causative R103H substitution in POLR3B, and to discover that riluzole, an FDA-approved drug for alleviation of ALS symptoms, partly corrects these assembly defects. Together, these results shed new light on the mechanism and regulation of human nuclear Pol III biogenesis.


Subject(s)
Neurodegenerative Diseases , RNA Polymerase III , Humans , RNA Polymerase III/genetics , Riluzole , DNA-Directed RNA Polymerases , Mutation, Missense
5.
J Proteome Res ; 21(4): 1073-1082, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35129352

ABSTRACT

The PAQosome (particle for arrangement of quaternary structure) is a 12-subunit HSP90 co-chaperone involved in the biogenesis of several human protein complexes. Two mechanisms of client selection have previously been identified, namely, the selective recruitment of specific adaptors and the differential use of homologous core subunits. Here, we describe a third client selection mechanism by showing that RPAP3, one of the core PAQosome subunits, is phosphorylated at several Ser residues in HEK293 cells. Affinity purification coupled with mass spectrometry (AP-MS) using the expression of tagged RPAP3 with single phospho-null mutations at Ser116, Ser119, or Ser121 reveals binding of the unphosphorylated form to several proteins involved in ribosome biogenesis. In vitro phosphorylation assays indicate that the kinase CK2 phosphorylates these RPAP3 residues. This finding is supported by data showing that pharmacological inhibition of CK2 enhances the binding of RPAP3 to ribosome preassembly factors in AP-MS experiments. Moreover, the silencing of PAQosome subunits interferes with ribosomal assembly factors' interactome. Altogether, these results indicate that RPAP3 phosphate group addition/removal at specific residues modulates binding to subunits of preribosomal complexes and allows speculating that PAQosome posttranslational modification is a mechanism of client selection.


Subject(s)
HSP90 Heat-Shock Proteins , Molecular Chaperones , HEK293 Cells , HSP90 Heat-Shock Proteins/metabolism , Humans , Molecular Chaperones/genetics , Ribosomal Proteins/genetics , Ribosomes/genetics , Ribosomes/metabolism
6.
Mol Cell ; 81(17): 3542-3559.e11, 2021 09 02.
Article in English | MEDLINE | ID: mdl-34380014

ABSTRACT

The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails, and binding partially disassembled nucleosomes. Here, we systematically test these and other scenarios in Saccharomyces cerevisiae and find that FACT binds transcribed chromatin, not RNAPII. Through a combination of high-resolution genome-wide mapping, single-molecule tracking, and mathematical modeling, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.


Subject(s)
DNA-Binding Proteins/metabolism , High Mobility Group Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription, Genetic/genetics , Transcriptional Elongation Factors/metabolism , Chromatin/metabolism , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins/genetics , High Mobility Group Proteins/genetics , Histone Chaperones/genetics , Histones/genetics , Histones/metabolism , Molecular Chaperones/metabolism , Nucleosomes/metabolism , Protein Binding , RNA Polymerase II/metabolism , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Transcriptional Elongation Factors/genetics
7.
HGG Adv ; 2(3): 100034, 2021 Jul 08.
Article in English | MEDLINE | ID: mdl-35047835

ABSTRACT

Leukodystrophies, genetic neurodevelopmental and/or neurodegenerative disorders of cerebral white matter, result from impaired myelin homeostasis and metabolism. Numerous genes have been implicated in these heterogeneous disorders; however, many individuals remain without a molecular diagnosis. Using whole-exome sequencing, biallelic variants in LSM7 were uncovered in two unrelated individuals, one with a leukodystrophy and the other who died in utero. LSM7 is part of the two principle LSM protein complexes in eukaryotes, namely LSM1-7 and LSM2-8. Here, we investigate the molecular and functional outcomes of these LSM7 biallelic variants in vitro and in vivo. Affinity purification-mass spectrometry of the LSM7 variants showed defects in the assembly of both LSM complexes. Lsm7 knockdown in zebrafish led to central nervous system defects, including impaired oligodendrocyte development and motor behavior. Our findings demonstrate that variants in LSM7 cause misassembly of the LSM complexes, impair neurodevelopment of the zebrafish, and may be implicated in human disease. The identification of more affected individuals is needed before the molecular mechanisms of mRNA decay and splicing regulation are added to the categories of biological dysfunctions implicated in leukodystrophies, neurodevelopmental and/or neurodegenerative diseases.

8.
J Proteome Res ; 19(1): 18-27, 2020 01 03.
Article in English | MEDLINE | ID: mdl-31738558

ABSTRACT

The PAQosome is an 11-subunit chaperone involved in the biogenesis of several human protein complexes. We show that ASDURF, a recently discovered upstream open reading frame (uORF) in the 5' UTR of ASNSD1 mRNA, encodes the 12th subunit of the PAQosome. ASDURF displays significant structural homology to ß-prefoldins and assembles with the five known subunits of the prefoldin-like module of the PAQosome to form a heterohexameric prefoldin-like complex. A model of the PAQosome prefoldin-like module is presented. The data presented here provide an example of a eukaryotic uORF-encoded polypeptide whose function is not limited to cis-acting translational regulation of downstream coding sequence and highlights the importance of including alternative ORF products in proteomic studies.


Subject(s)
Molecular Chaperones , Proteomics , Humans , Molecular Chaperones/genetics , Open Reading Frames
9.
Cell Rep ; 28(5): 1206-1218.e8, 2019 07 30.
Article in English | MEDLINE | ID: mdl-31365865

ABSTRACT

Genomic DNA is framed by additional layers of information, referred to as the epigenome. Epigenomic marks such as DNA methylation, histone modifications, and histone variants are concentrated on specific genomic sites, where they can both instruct and reflect gene expression. How this information is maintained, notably in the face of transcription, is not completely understood. Specifically, the extent to which modified histones themselves are retained through RNA polymerase II passage is unclear. Here, we show that several histone modifications are mislocalized when the transcription-coupled histone chaperones FACT or Spt6 are disrupted in Saccharomyces cerevisiae. In the absence of functional FACT or Spt6, transcription generates nucleosome loss, which is partially compensated for by the increased activity of non-transcription-coupled histone chaperones. The random incorporation of transcription-evicted modified histones scrambles epigenomic information. Our work highlights the importance of local recycling of modified histones by FACT and Spt6 during transcription in the maintenance of the epigenomic landscape.


Subject(s)
DNA-Binding Proteins/metabolism , High Mobility Group Proteins/metabolism , Histone Chaperones/metabolism , Histones/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcriptional Elongation Factors/metabolism , DNA-Binding Proteins/genetics , High Mobility Group Proteins/genetics , Histone Chaperones/genetics , Histones/genetics , Nucleosomes/genetics , Nucleosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcriptional Elongation Factors/genetics
10.
Mol Brain ; 12(1): 59, 2019 06 20.
Article in English | MEDLINE | ID: mdl-31221184

ABSTRACT

Recessive mutations in the ubiquitously expressed POLR3A and POLR3B genes are the most common cause of POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), a rare childhood-onset disorder characterized by deficient cerebral myelin formation and cerebellar atrophy. POLR3A and POLR3B encode the two catalytic subunits of RNA Polymerase III (Pol III), which synthesizes numerous small non-coding RNAs. We recently reported that mice homozygous for the Polr3a mutation c.2015G > A (p.Gly672Glu) have no neurological abnormalities and thus do not recapitulate the human POLR3-HLD phenotype. To determine if other POLR3-HLD mutations can cause a leukodystrophy phenotype in mouse, we characterized mice carrying the Polr3b mutation c.308G > A (p.Arg103His). Surprisingly, homozygosity for this mutation was embryonically lethal with only wild-type and heterozygous animals detected at embryonic day 9.5. Using proteomics in a human cell line, we found that the POLR3B R103H mutation severely impairs assembly of the Pol III complex. We next generated Polr3aG672E/G672E/Polr3b+/R103Hdouble mutant mice but observed that this additional mutation was insufficient to elicit a neurological or transcriptional phenotype. Taken together with our previous study on Polr3a G672E mice, our results indicate that missense mutations in Polr3a and Polr3b can variably impair mouse development and Pol III function. Developing a proper model of POLR3-HLD is crucial to gain insights into the pathophysiological mechanisms involved in this devastating neurodegenerative disease.


Subject(s)
Embryo Loss/enzymology , Hereditary Central Nervous System Demyelinating Diseases/genetics , Mutation/genetics , RNA Polymerase III/genetics , Animals , Base Sequence , Embryo Loss/genetics , Gene Expression Regulation, Enzymologic , Gene Knock-In Techniques , HEK293 Cells , Hereditary Central Nervous System Demyelinating Diseases/physiopathology , Homozygote , Humans , Mice, Inbred C57BL , Mice, Mutant Strains , Motor Activity , Myelin Sheath/metabolism , RNA Polymerase III/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
11.
Mol Cell ; 73(4): 655-669.e7, 2019 02 21.
Article in English | MEDLINE | ID: mdl-30639244

ABSTRACT

In Saccharomyces cerevisiae, transcription termination at protein-coding genes is coupled to the cleavage of the nascent transcript, whereas most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3, and Sen1 (NNS). Termination involves RNA polymerase II CTD phosphorylation, but a systematic analysis of the contribution of individual residues would improve our understanding of the role of the CTD in this process. Here we investigated the effect of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this genes class. Instead, mutating Tyr1 led to widespread termination defects at non-coding genes terminating via NNS. Finally, we showed that Tyr1 is important for pausing in the 5' end of genes and that slowing down transcription suppresses termination defects. Our work highlights the importance of Tyr1-mediated pausing in NNS-dependent termination.


Subject(s)
DNA Helicases/metabolism , Nuclear Proteins/metabolism , RNA Helicases/metabolism , RNA Polymerase II/metabolism , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Transcription Termination, Genetic , Binding Sites , DNA Helicases/genetics , Gene Expression Regulation, Fungal , Mutation , Nuclear Proteins/genetics , Phosphorylation , Protein Binding , RNA Helicases/genetics , RNA Polymerase II/genetics , RNA-Binding Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Time Factors , Tyrosine
12.
Nat Commun ; 8: 15615, 2017 05 31.
Article in English | MEDLINE | ID: mdl-28561026

ABSTRACT

The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1-TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed.


Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Carrier Proteins/metabolism , DNA Helicases/metabolism , Phosphoproteins/metabolism , Ribonucleoprotein, U5 Small Nuclear/biosynthesis , Tumor Suppressor Proteins/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Alternative Splicing/genetics , Carrier Proteins/genetics , Cell Line , DNA Helicases/genetics , Energy Metabolism/genetics , HEK293 Cells , HeLa Cells , Humans , Phosphoproteins/genetics , RNA, Small Interfering/genetics , TOR Serine-Threonine Kinases/metabolism , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein
13.
Mol Brain ; 10(1): 13, 2017 04 13.
Article in English | MEDLINE | ID: mdl-28407788

ABSTRACT

Recessive mutations in the ubiquitously expressed POLR3A gene cause one of the most frequent forms of childhood-onset hypomyelinating leukodystrophy (HLD): POLR3-HLD. POLR3A encodes the largest subunit of RNA Polymerase III (Pol III), which is responsible for the transcription of transfer RNAs (tRNAs) and a large array of other small non-coding RNAs. In order to study the central nervous system pathophysiology of the disease, we introduced the French Canadian founder Polr3a mutation c.2015G > A (p.G672E) in mice, generating homozygous knock-in (KI/KI) as well as compound heterozygous mice for one Polr3a KI and one null allele (KI/KO). Both KI/KI and KI/KO mice are viable and are able to reproduce. To establish if they manifest a motor phenotype, WT, KI/KI and KI/KO mice were submitted to a battery of behavioral tests over one year. The KI/KI and KI/KO mice have overall normal balance, muscle strength and general locomotion. Cerebral and cerebellar Luxol Fast Blue staining and measurement of levels of myelin proteins showed no significant differences between the three groups, suggesting that myelination is not overtly impaired in Polr3a KI/KI and KI/KO mice. Finally, expression levels of several Pol III transcripts in the brain showed no statistically significant differences. We conclude that the first transgenic mice with a leukodystrophy-causing Polr3a mutation do not recapitulate the childhood-onset HLD observed in the majority of human patients with POLR3A mutations, and provide essential information to guide selection of Polr3a mutations for developing future mouse models of the disease.


Subject(s)
Hereditary Central Nervous System Demyelinating Diseases/genetics , Mutation/genetics , Myelin Sheath/metabolism , RNA Polymerase III/genetics , Animals , Cerebellum/pathology , Cerebellum/physiopathology , Gene Knock-In Techniques , Hereditary Central Nervous System Demyelinating Diseases/physiopathology , Homozygote , Humans , Mice, Inbred C57BL , Mice, Knockout , Motor Activity , Purkinje Cells/metabolism , Purkinje Cells/pathology , RNA Polymerase III/metabolism , Transcription, Genetic
14.
Methods Mol Biol ; 1334: 273-98, 2015.
Article in English | MEDLINE | ID: mdl-26404157

ABSTRACT

In the analysis of experimental data corresponding to the signal enrichment of chromatin features such as histone modifications throughout the genome, it is often useful to represent the signal over known regions of interest, such as genes, using aggregate or individual profiles. In the present chapter, we describe and explain the best practices on how to generate such profiles as well as other usages of the versatile aggregate profiler (VAP) tool (Coulombe et al., Nucleic Acids Res 42:W485-W493, 2014), with a particular focus on the new functionalities introduced in version 1.1.0 of VAP.


Subject(s)
Chromatin Immunoprecipitation/methods , Chromatin/genetics , Histone Code/genetics , Oligonucleotide Array Sequence Analysis/methods , Genome , Promoter Regions, Genetic
15.
Nat Commun ; 6: 7623, 2015 Jul 07.
Article in English | MEDLINE | ID: mdl-26151409

ABSTRACT

A small proportion of 4H (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) or RNA polymerase III (POLR3)-related leukodystrophy cases are negative for mutations in the previously identified causative genes POLR3A and POLR3B. Here we report eight of these cases carrying recessive mutations in POLR1C, a gene encoding a shared POLR1 and POLR3 subunit, also mutated in some Treacher Collins syndrome (TCS) cases. Using shotgun proteomics and ChIP sequencing, we demonstrate that leukodystrophy-causative mutations, but not TCS mutations, in POLR1C impair assembly and nuclear import of POLR3, but not POLR1, leading to decreased binding to POLR3 target genes. This study is the first to show that distinct mutations in a gene coding for a shared subunit of two RNA polymerases lead to selective modification of the enzymes' availability leading to two different clinical conditions and to shed some light on the pathophysiological mechanism of one of the most common hypomyelinating leukodystrophies, POLR3-related leukodystrophy.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Genes, Recessive , Genetic Predisposition to Disease , Hereditary Central Nervous System Demyelinating Diseases/genetics , RNA Polymerase III/metabolism , DNA-Directed RNA Polymerases/genetics , Gene Expression Regulation, Enzymologic/physiology , Homozygote , Humans , Mutation , RNA Polymerase III/genetics
16.
Nucleic Acids Res ; 42(Web Server issue): W485-93, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24753414

ABSTRACT

The analysis of genomic data such as ChIP-Seq usually involves representing the signal intensity level over genes or other genetic features. This is often illustrated as a curve (representing the aggregate profile of a group of genes) or as a heatmap (representing individual genes). However, no specific resource dedicated to easily generating such profiles is currently available. We therefore built the versatile aggregate profiler (VAP), designed to be used by experimental and computational biologists to generate profiles of genomic datasets over groups of regions of interest, using either an absolute or a relative method. Graphical representation of the results is automatically generated, and subgrouping can be performed easily, based on the orientation of the flanking annotations. The outputs include statistical measures to facilitate comparisons between groups or datasets. We show that, through its intuitive design and flexibility, VAP can help avoid misinterpretations of genomics data. VAP is highly efficient and designed to run on laptop computers by using a memory footprint control, but can also be easily compiled and run on servers. VAP is accessible at http://lab-jacques.recherche.usherbrooke.ca/vap/.


Subject(s)
Genomics/methods , Software , Internet
17.
Methods ; 48(4): 381-6, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19450687

ABSTRACT

Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory factors from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAPII transcription machinery, including a new protein assembly we named the RNAPII-Associated Protein 3 (RPAP3) complex.


Subject(s)
RNA Polymerase II/chemistry , RNA Polymerase II/physiology , Apoptosis Regulatory Proteins , Carrier Proteins/physiology , Chromatography, Liquid , Humans , Mass Spectrometry , Proteomics/methods , RNA Polymerase II/genetics
18.
Mol Cell ; 27(2): 262-274, 2007 Jul 20.
Article in English | MEDLINE | ID: mdl-17643375

ABSTRACT

We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.


Subject(s)
Nucleotidyltransferases/metabolism , Amino Acid Sequence , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Line , Humans , In Vitro Techniques , Macromolecular Substances , Molecular Sequence Data , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/genetics , Protein Interaction Mapping , RNA Interference , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , RNA Processing, Post-Transcriptional , Ribonucleoproteins, Small Nuclear/chemistry , Ribonucleoproteins, Small Nuclear/metabolism , Transcription, Genetic
19.
Nucleic Acids Res ; 35(Database issue): D122-6, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17148480

ABSTRACT

We describe PReMod, a new database of genome-wide cis-regulatory module (CRM) predictions for both the human and the mouse genomes. The prediction algorithm, described previously in Blanchette et al. (2006) Genome Res., 16, 656-668, exploits the fact that many known CRMs are made of clusters of phylogenetically conserved and repeated transcription factors (TF) binding sites. Contrary to other existing databases, PReMod is not restricted to modules located proximal to genes, but in fact mostly contains distal predicted CRMs (pCRMs). Through its web interface, PReMod allows users to (i) identify pCRMs around a gene of interest; (ii) identify pCRMs that have binding sites for a given TF (or a set of TFs) or (iii) download the entire dataset for local analyses. Queries can also be refined by filtering for specific chromosomal regions, for specific regions relative to genes or for the presence of CpG islands. The output includes information about the binding sites predicted within the selected pCRMs, and a graphical display of their distribution within the pCRMs. It also provides a visual depiction of the chromosomal context of the selected pCRMs in terms of neighboring pCRMs and genes, all of which are linked to the UCSC Genome Browser and the NCBI. PReMod: http://genomequebec.mcgill.ca/PReMod.


Subject(s)
Databases, Nucleic Acid , Regulatory Elements, Transcriptional , Transcription Factors/metabolism , Algorithms , Animals , Binding Sites , Genomics , Humans , Internet , Mice , User-Computer Interface
20.
Genome Res ; 16(5): 656-68, 2006 May.
Article in English | MEDLINE | ID: mdl-16606704

ABSTRACT

The identification of regulatory regions is one of the most important and challenging problems toward the functional annotation of the human genome. In higher eukaryotes, transcription-factor (TF) binding sites are often organized in clusters called cis-regulatory modules (CRM). While the prediction of individual TF-binding sites is a notoriously difficult problem, CRM prediction has proven to be somewhat more reliable. Starting from a set of predicted binding sites for more than 200 TF families documented in Transfac, we describe an algorithm relying on the principle that CRMs generally contain several phylogenetically conserved binding sites for a few different TFs. The method allows the prediction of more than 118,000 CRMs within the human genome. A subset of these is shown to be bound in vivo by TFs using ChIP-chip. Their analysis reveals, among other things, that CRM density varies widely across the genome, with CRM-rich regions often being located near genes encoding transcription factors involved in development. Predicted CRMs show a surprising enrichment near the 3' end of genes and in regions far from genes. We document the tendency for certain TFs to bind modules located in specific regions with respect to their target genes and identify TFs likely to be involved in tissue-specific regulation. The set of predicted CRMs, which is made available as a public database called PReMod (http://genomequebec.mcgill.ca/PReMod), will help analyze regulatory mechanisms in specific biological systems.


Subject(s)
Computational Biology , Gene Expression , Genome , Regulatory Sequences, Nucleic Acid , Transcription, Genetic , Algorithms , Chromosomes, Human , Computer Simulation , Humans , Physical Chromosome Mapping
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