ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans.

Background: Neural tube defects (NTDs) are caused by genetic and environmental factors. ARMC5 is part of a novel ubiquitin ligase specific for POLR2A, the largest subunit of RNA polymerase II (Pol II).

Results: We find that ARMC5 knockout mice have increased incidence of NTDs, such as spina bifida and exencephaly.

Variants in impair LSM complexes assembly, neurodevelopment in zebrafish and may be associated with an ultra-rare neurological disease.

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 were uncovered in two unrelated individuals, one with a leukodystrophy and the other who died .

Ser-Phosphorylation of PCSK9 (Proprotein Convertase Subtilisin-Kexin 9) by Fam20C (Family With Sequence Similarity 20, Member C) Kinase Enhances Its Ability to Degrade the LDLR (Low-Density Lipoprotein Receptor).

Objective: PCSK9 (proprotein convertase subtilisin-kexin 9) enhances the degradation of the LDLR (low-density lipoprotein receptor) in endosomes/lysosomes. This study aimed to determine the sites of PCSK9 phosphorylation at Ser-residues and the consequences of such posttranslational modification on the secretion and activity of PCSK9 on the LDLR. Approach and Results: Fam20C (family with sequence similarity 20, member C) phosphorylates serines in secretory proteins containing the motif S-X-E/phospho-Ser, including the cholesterol-regulating PCSK9.

Leukodystrophy-associated mutations down-regulate the RNA polymerase III transcript and important regulatory RNA .

RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in , encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive.

Bi-allelic Mutations in EPRS, Encoding the Glutamyl-Prolyl-Aminoacyl-tRNA Synthetase, Cause a Hypomyelinating Leukodystrophy.

Hypomyelinating leukodystrophies are genetic disorders characterized by insufficient myelin deposition during development. They are diagnosed on the basis of both clinical and MRI features followed by genetic confirmation. Here, we report on four unrelated affected individuals with hypomyelination and bi-allelic pathogenic variants in EPRS, the gene encoding cytoplasmic glutamyl-prolyl-aminoacyl-tRNA synthetase.

Absence of neurological abnormalities in mice homozygous for the Polr3a G672E hypomyelinating leukodystrophy mutation.

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.

Delayed Reconstruction by Total Calcaneal Allograft following Calcanectomy: Is It an Option?

Many options are available in literature for the management of delayed reconstruction following calcanectomy. In cases of low-grade tumor lesions, conservative surgery can be considered. We describe a case of delayed reconstruction by calcaneal allograft after calcanectomy for low-grade chondrosarcoma.

Recessive mutations in POLR1C cause a leukodystrophy by impairing biogenesis of RNA polymerase III.

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.

Abnormal expression of RNA polymerase II-associated proteins in muscle of patients with myofibrillar myopathies.

Aims: Myofibrillar myopathies (MFMs) are a group of inherited or sporadic neuromuscular disorders characterized morphologically by foci of myofibril dissolution, disintegration of the Z-disk and insoluble protein aggregates within the muscle fibres. The sequential events leading to muscle fibre damage remains largely unknown.

Methods And Results: We investigated the expression and the cellular localization of RNA polymerase II (RNAPII)-associated proteins (RPAPs) in muscle biopsies from patients with genetically proven and sporadic MFMs.

Nuclear import of RNA polymerase II is coupled with nucleocytoplasmic shuttling of the RNA polymerase II-associated protein 2.

The RNA polymerase II (RNAP II)-associated protein (RPAP) 2 has been discovered through its association with various subunits of RNAP II in affinity purification coupled with mass spectrometry experiments. Here, we show that RPAP2 is a mainly cytoplasmic protein that shuttles between the cytoplasm and the nucleus. RPAP2 shuttling is tightly coupled with nuclear import of RNAP II, as RPAP2 silencing provokes abnormal accumulation of RNAP II in the cytoplasmic space.

Discovery of cell compartment specific protein-protein interactions using affinity purification combined with tandem mass spectrometry.

Affinity purification combined with tandem mass spectrometry (AP-MS/MS) is a well-established method used to discover interaction partners for a given protein of interest. Because most AP-MS/MS approaches are performed using the soluble fraction of whole cell extracts (WCE), information about the cellular compartments where the interactions occur is lost. More importantly, classical AP-MS/MS often fails to identify interactions that take place in the nonsoluble fraction of the cell, for example, on the chromatin or membranes; consequently, protein complexes that are less soluble are underrepresented.

The role of the spontaneous and evoked k-complex in good-sleeper controls and in individuals with insomnia.

Study Objectives: Spontaneous and evoked K-complexes are believed to reflect a mechanism of sleep protection, yet their functional role remains a subject of debate. Studying the K-complex in primary insomnia, for which sleep protection appears to be of central importance, may provide further insight on its role. The purpose of the present study was to determine the role of spontaneous and evoked K-complexes in good sleepers and to investigate its role in primary insomnia.

The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II.

RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts.

An extensive study of the genetic diversity within seven French wine grape variety collections.

The process of vegetative propagation used to multiply grapevine varieties produces, in most cases, clones genetically identical to the parental plant. Nevertheless, spontaneous somatic mutations can occur in the regenerative cells that give rise to the clones, leading to consider varieties as populations of clones that conform to a panel of phenotypic traits. Using two sets of nuclear microsatellite markers, the present work aimed at evaluating and comparing the intravarietal genetic diversity within seven wine grape varieties: Cabernet franc, Cabernet Sauvignon, Chenin blanc, Grolleau, Pinot noir, Riesling, Savagnin, comprising a total number of 344 accessions of certified clones and introductions preserved in French repositories.

Spontaneous K-complexes in chronic psychophysiological insomnia.

Objective: Spontaneous K-complexes are electroencephalographic features unique to non-rapid eye movement sleep. It has been suggested that this phasic event is a sleep-protective mechanism. Because insomnia sufferers report poor sleep quantity and quality, the objective of this study was to document the occurrence of spontaneous K-complexes in Stage 2 sleep of individuals with chronic insomnia.

High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes.

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.

Use of site-specific protein-DNA photocrosslinking of purified complexes to analyze the topology of the RNA polymerase II transcription initiation complex.

A method for the photocrosslinking of proteins to DNA in purified complexes is described. It makes use of the juxtaposition of a limited number of photoreactive nucleotides with a limited number of radiolabeled nucleotides at a specific location in a DNA fragment. Protein-DNA complexes are submitted to an electrophoretic mobility shift assay that is then irradiated with UV light in order to crosslink the proteins to DNA.

Genomic location of the human RNA polymerase II general machinery: evidence for a role of TFIIF and Rpb7 at both early and late stages of transcription.

The functions ascribed to the mammalian GTFs (general transcription factors) during the various stages of the RNAPII (RNA polymerase II) transcription reaction are based largely on in vitro studies. To gain insight as to the functions of the GTFs in living cells, we have analysed the genomic location of several human GTF and RNAPII subunits carrying a TAP (tandem-affinity purification) tag. ChIP (chromatin immunoprecipitation) experiments using anti-tag beads (TAP-ChIP) allowed the systematic localization of the tagged factors.

Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

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.

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo.

Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors.

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position -3 to +6.

Photo-cross-linking of a purified preinitiation complex reveals central roles for the RNA polymerase II mobile clamp and TFIIE in initiation mechanisms.

The topological organization of a TATA binding protein-TFIIB-TFIIF-RNA polymerase II (RNAP II)-TFIIE-promoter complex was analyzed using site-specific protein-DNA photo-cross-linking of gel-purified complexes. The cross-linking results for the subunits of RNAP II were used to determine the path of promoter DNA against the structure of the enzyme. The results indicate that promoter DNA wraps around the mobile clamp of RNAP II.