Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

• MeSH
• 5' Untranslated Regions MeSH
• Eukaryotic Initiation Factor-2 genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Peptide Initiation Factors genetics   metabolism   MeSH
• Codon, Initiator MeSH
• Humans MeSH
• Ribosome Subunits, Small, Eukaryotic genetics   metabolism   MeSH
• Multiprotein Complexes genetics   metabolism   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Activating Transcription Factor 4 genetics   metabolism   MeSH
• Basic-Leucine Zipper Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A family of Structural Maintenance of Chromosome (SMC) complexes is essential for key cellular processes ensuring proper cohesion, condensation and replication. They share a common SMC-kleisin architecture allowing them to embrace DNA. In SMC5/6, the NSE1 and NSE3 KITE and NSE4 kleisin subunits form a stable subcomplex that binds DNA and regulates essential processes. In addition, NSE5 and NSE6 subunits associate with the core SMC5/6 complex and recruit it to DNA repair sites. The architecture of the SMC5/6 complex is crucial for its proper functioning, and mutations within the human SMC5/6 subunits result in severe syndromes. Therefore, we aimed to analyze interactions within the human SMC5/6 complex and determine its detailed architecture. Firstly, we analyzed different parts of SMC5/6 by crosslinking and MS/MS analysis. Our data suggested domain arrangements of hNSE1-hNSE3 and orientation of hNSE4 within the hNSE1-hNSE3-hNSE4 subcomplex. The crosslinking and electron microscopic analysis of the SMC5/6 core complex showed its rod-like architecture with juxtaposed hSMC5-hSMC6 arms. Additionally, we observed fully or partially opened hSMC5-hSMC6 shapes with the hNSE1-hNSE3-hNSE4 trimer localized in the SMC head domains. To complete mapping of the human SMC5/6 complex architecture, we analyzed positions of hNSE5-hNSE6 at the hSMC5-hSMC6 arms. We showed that hNSE6 binding to hNSE5 and the coiled-coil arm of hSMC6 is mediated by a conserved FAM178 domain, which we therefore renamed CANIN (Coiled-coil SMC6 And NSE5 INteracting) domain. Interestingly, hNSE6 bound both hSMC5 and hSMC6 arms, suggesting that hNSE6 may lock the arms and regulate the dynamics of the human SMC5/6 complex.

• MeSH
• Chromosomal Proteins, Non-Histone genetics   MeSH
• Humans MeSH
• Protein Multimerization genetics   MeSH
• Multiprotein Complexes genetics   MeSH
• Mutation MeSH
• DNA Repair genetics   MeSH
• Protein Domains genetics   MeSH
• Cell Cycle Proteins genetics   MeSH
• Carrier Proteins genetics   MeSH
• Protein Binding genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In the extremophile bacterium Deinococcus radiodurans, the outermost surface layer is tightly connected with the rest of the cell wall. This integrated organization provides a compact structure that shields the bacterium against environmental stresses. The fundamental unit of this surface layer (S-layer) is the S-layer deinoxanthin-binding complex (SDBC), which binds the carotenoid deinoxanthin and provides both, thermostability and UV radiation resistance. However, the structural organization of the SDBC awaits elucidation. Here, we report the isolation of the SDBC with a gentle procedure consisting of lysozyme treatment and solubilization with the nonionic detergent n-dodecyl-β-d-maltoside, which preserved both hydrophilic and hydrophobic components of the SDBC and allows the retention of several minor subunits. As observed by low-resolution single-particle analysis, we show that the complex possesses a porin-like structural organization, but is larger than other known porins. We also noted that the main SDBC component, the protein DR_2577, shares regions of similarity with known porins. Moreover, results from electrophysiological assays with membrane-reconstituted SDBC disclosed that it is a nonselective channel that has some peculiar gating properties, but also exhibits behavior typically observed in pore-forming proteins, such as porins and ionic transporters. The functional properties of this system and its porin-like organization provide information critical for understanding ion permeability through the outer cell surface of S-layer-carrying bacterial species.

• MeSH
• Bacterial Proteins chemistry   genetics   MeSH
• Cell Membrane chemistry   MeSH
• Cell Wall chemistry   MeSH
• Deinococcus chemistry   genetics   MeSH
• Carotenoids chemistry   MeSH
• Membrane Glycoproteins chemistry   MeSH
• Multiprotein Complexes chemistry   genetics   MeSH
• Porins chemistry   MeSH
• Protein Binding genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

PURPOSE: To define the phenotypic and mutational spectrum of epilepsies related to DEPDC5, NPRL2 and NPRL3 genes encoding the GATOR1 complex, a negative regulator of the mTORC1 pathway METHODS: We analyzed clinical and genetic data of 73 novel probands (familial and sporadic) with epilepsy-related variants in GATOR1-encoding genes and proposed new guidelines for clinical interpretation of GATOR1 variants. RESULTS: The GATOR1 seizure phenotype consisted mostly in focal seizures (e.g., hypermotor or frontal lobe seizures in 50%), with a mean age at onset of 4.4 years, often sleep-related and drug-resistant (54%), and associated with focal cortical dysplasia (20%). Infantile spasms were reported in 10% of the probands. Sudden unexpected death in epilepsy (SUDEP) occurred in 10% of the families. Novel classification framework of all 140 epilepsy-related GATOR1 variants (including the variants of this study) revealed that 68% are loss-of-function pathogenic, 14% are likely pathogenic, 15% are variants of uncertain significance and 3% are likely benign. CONCLUSION: Our data emphasize the increasingly important role of GATOR1 genes in the pathogenesis of focal epilepsies (>180 probands to date). The GATOR1 phenotypic spectrum ranges from sporadic early-onset epilepsies with cognitive impairment comorbidities to familial focal epilepsies, and SUDEP.

• MeSH
• Brugada Syndrome genetics   mortality   physiopathology   MeSH
• Child MeSH
• Epilepsy complications   epidemiology   genetics   physiopathology   MeSH
• Genetic Predisposition to Disease MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Mechanistic Target of Rapamycin Complex 1 genetics   MeSH
• Multiprotein Complexes genetics   MeSH
• INDEL Mutation genetics   MeSH
• Loss of Function Mutation genetics   MeSH
• Tumor Suppressor Proteins genetics   MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• GTPase-Activating Proteins genetics   MeSH
• Repressor Proteins genetics   MeSH
• Pedigree MeSH
• Signal Transduction genetics   MeSH
• DNA Copy Number Variations genetics   MeSH
• Seizures complications   epidemiology   genetics   physiopathology   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.

• MeSH
• Chromosomes genetics   MeSH
• DNA-Binding Proteins chemistry   genetics   MeSH
• DNA Topoisomerases, Type II chemistry   genetics   MeSH
• DNA chemistry   genetics   MeSH
• DNA Breaks, Double-Stranded * MeSH
• Etoposide chemistry   MeSH
• Transcription, Genetic MeSH
• Gene Conversion genetics   MeSH
• HCT116 Cells MeSH
• Topoisomerase II Inhibitors chemistry   pharmacology   MeSH
• Kinetics MeSH
• Humans MeSH
• Multiprotein Complexes chemistry   genetics   MeSH
• DNA Repair genetics   MeSH
• Poly-ADP-Ribose Binding Proteins chemistry   genetics   MeSH
• Torsion, Mechanical MeSH
• Translocation, Genetic genetics   MeSH
• Chromosome Breakage MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

C-terminal mutations of the nucleolar protein nucleophosmin (NPM) are the most frequent genetic aberration detected in acute myeloid leukemia (AML) with normal karyotype. The mutations cause aberrant cytoplasmic localization of NPM and lead to loss of functions associated with NPM nucleolar localization, e.g. in ribosome biogenesis or DNA-damage repair. NPM has many interaction partners and some of them were proved to interact also with the mutated form (NPMmut) and due to this interaction thereby to be withdrawn from their site of action. We analyzed the impact of the mutation on NPM interaction with nucleolin (NCL) which is also prevalently localized into the nucleolus and cooperates with wild-type NPM (NPMwt) in many cellular processes. We revealed that the NCL-NPM complex formation is completely abolished by the mutation and that the presence/absence of the interaction is not affected by drugs causing genotoxic stress or differentiation. Deregulation resulting from changes of NCL/NPMwt ratio may contribute to leukemogenesis.

• MeSH
• Leukemia, Myeloid, Acute genetics   metabolism   pathology   MeSH
• Cell Nucleolus genetics   metabolism   pathology   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Multiprotein Complexes genetics   metabolism   MeSH
• Mutation * MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins genetics   metabolism   MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein-protein and protein-DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.

• MeSH
• Biophysical Phenomena MeSH
• DNA-Binding Proteins chemistry   genetics   MeSH
• Escherichia coli chemistry   genetics   MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Multiprotein Complexes chemistry   genetics   MeSH
• Protein Subunits chemistry   genetics   MeSH
• Protein Domains genetics   MeSH
• Escherichia coli Proteins chemistry   genetics   MeSH
• Deoxyribonucleases, Type I Site-Specific chemistry   genetics   MeSH
• Amino Acid Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We describe a novel mechanism of mRNA decay regulation, which takes place under the conditions of glucose deprivation in the yeast Saccharomyces cerevisiae. The regulation is based on temporally stable sequestration of the main 5'-3' mRNA exoribonuclease Xrn1 at the eisosome, a plasma membrane-associated protein complex organizing a specialized membrane microdomain. As documented by monitoring the decay of a specific mRNA substrate in time, Xrn1-mediated mRNA degradation ceases during the accumulation of Xrn1 at eisosome, but the eisosome-associated Xrn1 retains its functionality and can be re-activated when released to cytoplasm following the addition of glucose. In cells lacking the eisosome organizer Pil1, Xrn1 does not associate with the plasma membrane and its activity is preserved till the stationary phase. Thus, properly assembled eisosome is necessary for this kind of Xrn1 regulation, which occurs in a liquid culture as well as in a differentiated colony.

• MeSH
• Cell Membrane genetics   metabolism   MeSH
• Cytoplasm genetics   metabolism   MeSH
• Exoribonucleases genetics   metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Membrane Microdomains genetics   metabolism   MeSH
• Membrane Proteins genetics   metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Multiprotein Complexes genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• RNA Stability genetics   MeSH
• Publication type
• Journal Article MeSH

One recently discussed general mechanism affecting gene expression is 3'-untranslated region (3'UTR) length. Events such as shortening, translocation or loss of 3'UTRs are observed within oncogenes and are proposed to associate with increased expression. Thus, increased efforts are being made to understand constitutive and differential transcript 3'end formation. Investigation of AGR2 mRNA revealed a direct impact of its 3'UTR length on AGR2 expression. In silico analyses identified several regulatory sequences within the distal part of AGR2 mRNA that may regulate 3'UTR length and associated protein levels. Short 3'UTRs were observed in a panel of AGR2-positive cancer cell lines and in human breast cancer specimens, in which more extensive 3'UTR shortening correlated with increased AGR2 protein levels. AGR2 is an important member of PI3K/AKT signalling pathway, which along with the proposed involvement of mTOR in the regulation of alternative polyadenylation, prompted us to study the role of mTOR in relation to AGR2 mRNA 3'UTR shortening. A direct impact of mTOR signalling on AGR2 3'UTR shortening associated with increased protein synthesis was found, which led to the identification of a novel molecular mechanism involved in upregulation of AGR2 levels in mTOR-activated cells via modulating the 3'UTR length of AGR2 mRNA.

• MeSH
• 3' Untranslated Regions genetics   MeSH
• A549 Cells MeSH
• HCT116 Cells MeSH
• HEK293 Cells MeSH
• Cloning, Molecular MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• MCF-7 Cells MeSH
• Multiprotein Complexes genetics   MeSH
• Cell Line, Tumor MeSH
• Breast Neoplasms genetics   MeSH
• Polyadenylation genetics   MeSH
• Proteins genetics   metabolism   MeSH
• Protein Biosynthesis genetics   MeSH
• Gene Expression Regulation genetics   MeSH
• Regulatory Sequences, Ribonucleic Acid genetics   MeSH
• Signal Transduction genetics   MeSH
• TOR Serine-Threonine Kinases genetics   metabolism   MeSH
• Up-Regulation genetics   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal β-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a "core" DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.

• MeSH
• Cell Differentiation genetics   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• DNA (Cytosine-5-)-Methyltransferases genetics   metabolism   MeSH
• DNA (Cytosine-5-)-Methyltransferase 1 MeSH
• Erythroid Cells chemistry   metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• DNA Methylation genetics   MeSH
• Multiprotein Complexes genetics   metabolism   MeSH
• Mice MeSH
• Proto-Oncogene Proteins genetics   metabolism   MeSH
• Gene Expression Regulation genetics   MeSH
• Repressor Proteins genetics   metabolism   MeSH
• Trans-Activators genetics   metabolism   MeSH
• GATA1 Transcription Factor genetics   metabolism   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH