Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 63
Filter
1.
Proc Natl Acad Sci U S A ; 120(39): e2305756120, 2023 09 26.
Article in English | MEDLINE | ID: mdl-37722062

ABSTRACT

Mutations in RNA/DNA-binding proteins cause amyotrophic lateral sclerosis (ALS), but the underlying disease mechanisms remain unclear. Here, we report that a set of ALS-associated proteins, namely FUS, EWSR1, TAF15, and MATR3, impact the expression of genes encoding the major histocompatibility complex II (MHC II) antigen presentation pathway. Both subunits of the MHC II heterodimer, HLA-DR, are down-regulated in ALS gene knockouts/knockdown in HeLa and human microglial cells, due to loss of the MHC II transcription factor CIITA. Importantly, hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells bearing the FUSR495X mutation and HPCs derived from C9ORF72 ALS patient induced pluripotent stem cells also exhibit disrupted MHC II expression. Given that HPCs give rise to numerous immune cells, our data raise the possibility that loss of the MHC II pathway results in global failure of the immune system to protect motor neurons from damage that leads to ALS.


Subject(s)
Amyotrophic Lateral Sclerosis , Humans , Amyotrophic Lateral Sclerosis/genetics , Antigen Presentation/genetics , Genes, MHC Class II , Major Histocompatibility Complex , Motor Neurons , RNA-Binding Proteins/genetics , Nuclear Matrix-Associated Proteins
2.
Psychiatr Serv ; 71(6): 627-630, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32041510

ABSTRACT

Evidence-based depression treatment in primary care is well established. However, clinicians are less likely to be trained to diagnose and treat anxiety disorder, which is frequently comorbid, poses an independent risk for suicidality, and complicates disease management. The University of North Carolina's Internal Medicine Clinic developed a measurement-guided approach to identifying and treating anxiety disorder using the seven-item Generalized Anxiety Disorder Scale, treatment algorithms, medication charts, case-based training for best practices, onsite behavioral counseling, and psychiatric consultation. NAMASTE (new anxiety management algorithm standardizing treatment experience) offers a treatment approach for primary care and addresses a major unmet need in public health and medical education.


Subject(s)
Anxiety Disorders/therapy , Patient Care Management/organization & administration , Primary Health Care/standards , Algorithms , Cognitive Behavioral Therapy , Humans
3.
Proc Natl Acad Sci U S A ; 116(16): 7837-7846, 2019 04 16.
Article in English | MEDLINE | ID: mdl-30923118

ABSTRACT

To ensure efficient and accurate gene expression, pre-mRNA processing and mRNA export need to be balanced. However, how this balance is ensured remains largely unclear. Here, we found that SF3b, a component of U2 snRNP that participates in splicing and 3' processing of pre-mRNAs, interacts with the key mRNA export adaptor THO in vivo and in vitro. Depletion of SF3b reduces THO binding with the mRNA and causes nuclear mRNA retention. Consistently, introducing SF3b binding sites into the mRNA enhances THO recruitment and nuclear export in a dose-dependent manner. These data demonstrate a role of SF3b in promoting mRNA export. In support of this role, SF3b binds with mature mRNAs in the cells. Intriguingly, disruption of U2 snRNP by using a U2 antisense morpholino oligonucleotide does not inhibit, but promotes, the role of SF3b in mRNA export as a result of enhanced SF3b-THO interaction and THO recruitment to the mRNA. Together, our study uncovers a U2-snRNP-independent role of SF3b in mRNA export and suggests that SF3b contributes to balancing pre-mRNA processing and mRNA export.


Subject(s)
Phosphoproteins , RNA Precursors , RNA Splicing Factors , RNA, Messenger , Ribonucleoprotein, U2 Small Nuclear , Binding Sites/genetics , HeLa Cells , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing/genetics , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribonucleoprotein, U2 Small Nuclear/genetics , Ribonucleoprotein, U2 Small Nuclear/metabolism
4.
Cancer Cell ; 35(2): 283-296.e5, 2019 02 11.
Article in English | MEDLINE | ID: mdl-30712845

ABSTRACT

SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment.


Subject(s)
B-Lymphocytes/immunology , Cellular Senescence , Gene Deletion , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mutation , Neoplasms, Experimental/genetics , Phosphoproteins/genetics , RNA Splicing Factors/genetics , Receptors, Antigen, B-Cell/immunology , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Agammaglobulinaemia Tyrosine Kinase/metabolism , Alternative Splicing , Animals , Antineoplastic Agents/pharmacology , Ataxia Telangiectasia Mutated Proteins/deficiency , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Cellular Senescence/drug effects , DNA Damage , Genetic Predisposition to Disease , Genomic Instability , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/immunology , Neoplasms, Experimental/metabolism , Phenotype , Phosphoproteins/metabolism , Piperidines , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA Splicing Factors/metabolism , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Tumor Cells, Cultured
5.
Nucleic Acids Res ; 46(22): 11939-11951, 2018 12 14.
Article in English | MEDLINE | ID: mdl-30398641

ABSTRACT

Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Muscular Atrophy, Spinal/genetics , Nuclear Matrix-Associated Proteins/genetics , RNA-Binding Protein EWS/genetics , RNA-Binding Protein FUS/genetics , RNA-Binding Proteins/genetics , TATA-Binding Protein Associated Factors/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , CRISPR-Cas Systems , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , Gene Editing , Gene Expression Regulation , Gene Knockout Techniques , Humans , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/pathology , Nuclear Matrix-Associated Proteins/deficiency , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Positive Transcriptional Elongation Factor B/genetics , Positive Transcriptional Elongation Factor B/metabolism , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Transfer/genetics , RNA, Transfer/metabolism , RNA-Binding Protein EWS/deficiency , RNA-Binding Protein FUS/deficiency , Ribonucleoprotein, U1 Small Nuclear/genetics , Ribonucleoprotein, U1 Small Nuclear/metabolism , Spliceosomes/chemistry , Spliceosomes/metabolism , TATA-Binding Protein Associated Factors/deficiency , Transcription Factors, TFII/genetics , Transcription Factors, TFII/metabolism
6.
JCI Insight ; 3(19)2018 10 04.
Article in English | MEDLINE | ID: mdl-30282833

ABSTRACT

The identification of targetable vulnerabilities in the context of therapeutic resistance is a key challenge in cancer treatment. We detected pervasive aberrant splicing as a characteristic feature of chronic lymphocytic leukemia (CLL), irrespective of splicing factor mutation status, which was associated with sensitivity to the spliceosome modulator, E7107. Splicing modulation affected CLL survival pathways, including members of the B cell lymphoma-2 (BCL2) family of proteins, remodeling antiapoptotic dependencies of human and murine CLL cells. E7107 treatment decreased myeloid cell leukemia-1 (MCL1) dependence and increased BCL2 dependence, sensitizing primary human CLL cells and venetoclax-resistant CLL-like cells from an Eµ-TCL1-based adoptive transfer murine model to treatment with the BCL2 inhibitor venetoclax. Our data provide preclinical rationale to support the combination of venetoclax with splicing modulators to reprogram apoptotic dependencies in CLL for treating venetoclax-resistant CLL cases.


Subject(s)
Alternative Splicing/drug effects , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Epoxy Compounds/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Macrolides/pharmacology , Sulfonamides/pharmacology , Adult , Aged , Aged, 80 and over , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Apoptosis/genetics , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Drug Screening Assays, Antitumor , Epoxy Compounds/therapeutic use , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Macrolides/therapeutic use , Male , Mice , Mice, Transgenic , Middle Aged , Mitochondria/drug effects , Mitochondria/pathology , Mutation , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Phosphoproteins/genetics , Primary Cell Culture , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , RNA Splicing Factors/genetics , Spliceosomes/drug effects , Spliceosomes/metabolism , Sulfonamides/therapeutic use , Thiophenes/pharmacology , Thiophenes/therapeutic use
7.
Methods Mol Biol ; 1823: 43-50, 2018.
Article in English | MEDLINE | ID: mdl-29959672

ABSTRACT

In the genome, primary microRNAs (pri-miRNAs) are encoded either as independent transcriptional units with their own promoters (intergenic miRNAs) or within the introns of other genes (intronic miRNAs). Here, we report two methods, one that we established for coupled RNAP II transcription and pri-miRNA processing and the other that is a three-way system for RNAP II transcription, pri-miRNA processing, and pre-mRNA splicing. In these systems, CMV-DNA constructs encoding the processing substrates are incubated in HeLa cell nuclear extracts in the presence of 32P-UTP to generate the nascent RNAP II transcripts, which are processed efficiently by the endogenous RNA processing machineries in nuclear extracts.


Subject(s)
MicroRNAs/biosynthesis , RNA Polymerase II/chemistry , RNA Precursors/biosynthesis , RNA Splicing , Transcription, Genetic , Cell-Free System/chemistry , HeLa Cells , Humans , MicroRNAs/chemistry , RNA Precursors/chemistry
8.
Sci Rep ; 8(1): 8755, 2018 06 08.
Article in English | MEDLINE | ID: mdl-29884807

ABSTRACT

Mutations in multiple RNA/DNA binding proteins cause Amyotrophic Lateral Sclerosis (ALS). Included among these are the three members of the FET family (FUS, EWSR1 and TAF15) and the structurally similar MATR3. Here, we characterized the interactomes of these four proteins, revealing that they largely have unique interactors, but share in common an association with U1 snRNP. The latter observation led us to analyze the interactome of the U1 snRNP machinery. Surprisingly, this analysis revealed the interactome contains ~220 components, and of these, >200 are shared with the RNA polymerase II (RNAP II) machinery. Among the shared components are multiple ALS and Spinal muscular Atrophy (SMA)-causative proteins and numerous discrete complexes, including the SMN complex, transcription factor complexes, and RNA processing complexes. Together, our data indicate that the RNAP II/U1 snRNP machinery functions in a wide variety of molecular pathways, and these pathways are candidates for playing roles in ALS/SMA pathogenesis.


Subject(s)
Nuclear Matrix-Associated Proteins/metabolism , Protein Interaction Maps , RNA Polymerase II/metabolism , RNA-Binding Protein EWS/metabolism , RNA-Binding Protein FUS/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/metabolism , TATA-Binding Protein Associated Factors/metabolism , Amyotrophic Lateral Sclerosis/metabolism , HeLa Cells , Humans
9.
J Nephrol ; 31(1): 157-164, 2018 02.
Article in English | MEDLINE | ID: mdl-27848227

ABSTRACT

The etiology and treatment of transplant glomerulopathy (TG) is not clear. TG is associated with donor-specific antibodies but the lack of C4d deposition in the peritubular capillaries (ptc-C4d) in some cases has caused the role of complement in the pathogenesis of TG to be debated. There is however, little information on C4d deposition in the glomerulus itself. We retrieved random frozen sections from 25 cases with well-established TG by light microscopy (LM) and 25 cases without TG as controls and reviewed the LM and immunofluorescence (nine biopsies were excluded due to inadequate samples). Glomerular complement deposition was assessed in all included biopsies. Glomerular C3d and C4d deposition occurred in a distinct pattern in all TG biopsies: segmental or global double linear staining of the glomerular capillary wall in 23 (100%). This pattern was not present in any NON-TG biopsies. The distinct glomerular complement deposition patterns in all TG cases are suggestive that TG is a proximal complement-mediated process and therapies should focus on proximal complement inhibition.


Subject(s)
Complement C3d/analysis , Complement C4b/analysis , Kidney Diseases/immunology , Kidney Glomerulus/immunology , Kidney Transplantation/adverse effects , Peptide Fragments/analysis , Biomarkers/analysis , Case-Control Studies , Complement Activation , Fluorescent Antibody Technique , Frozen Sections , Humans , Kidney Diseases/diagnosis , Kidney Glomerulus/pathology
10.
Cell Rep ; 19(11): 2244-2256, 2017 06 13.
Article in English | MEDLINE | ID: mdl-28614712

ABSTRACT

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , C9orf72 Protein/metabolism , Dipeptides/pharmacology , Frontotemporal Dementia/genetics , RNA, Small Nuclear/metabolism , Amyotrophic Lateral Sclerosis/immunology , Amyotrophic Lateral Sclerosis/metabolism , C9orf72 Protein/genetics , DNA Repeat Expansion , Dipeptides/metabolism , Frontotemporal Dementia/immunology , Frontotemporal Dementia/metabolism , Humans , Proteomics/methods , RNA Splicing , RNA, Small Nuclear/genetics , Ribonucleoprotein, U2 Small Nuclear/genetics , Ribonucleoprotein, U2 Small Nuclear/metabolism
11.
Elife ; 62017 02 08.
Article in English | MEDLINE | ID: mdl-28177281

ABSTRACT

Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent. One of these, the pre-mRNA splicing factor SF3B1, is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency.


Subject(s)
Gene Dosage , Neoplasms/genetics , Neoplasms/pathology , Phosphoproteins/genetics , RNA Splicing Factors/genetics , Cell Line, Tumor , Humans
12.
Cancer Cell ; 30(5): 750-763, 2016 Nov 14.
Article in English | MEDLINE | ID: mdl-27818134

ABSTRACT

Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch signaling (mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively). SF3B1 mutation leads to diverse changes in CLL-related pathways.


Subject(s)
Alternative Splicing , Gene Expression Profiling/methods , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mutation , Phosphoproteins/genetics , RNA Splicing Factors/genetics , Cell Line, Tumor , Dishevelled Proteins/genetics , Gene Expression Regulation, Neoplastic , Humans , Receptors, Notch/genetics , Signal Transduction
13.
Cell Discov ; 12015.
Article in English | MEDLINE | ID: mdl-26491543

ABSTRACT

mRNAs containing premature termination codons (PTCs) are known to be degraded via nonsense-mediated mRNA decay (NMD). Unexpectedly, we found that mRNAs containing any type of PTC (UAA, UAG, UGA) are detained in the nucleus whereas their wild-type counterparts are rapidly exported. This retention is strictly reading-frame dependent. Strikingly, our data indicate that translating ribosomes in the nucleus proofread the frame and detect the PTCs in the nucleus. Moreover, the shuttling NMD protein Upf1 specifically associates with PTC+ mRNA in the nucleus and is required for nuclear retention of PTC+ mRNA. Together, our data lead to a working model that PTCs are recognized in the nucleus by translating ribosomes, resulting in recruitment of Upf1, which in turn functions in nuclear retention of PTC+ mRNA. Nuclear PTC recognition adds a new layer of proofreading for mRNA and may be vital for ensuring the extraordinary fidelity required for protein production.

14.
Sci Rep ; 5: 11992, 2015 Jul 07.
Article in English | MEDLINE | ID: mdl-26149087

ABSTRACT

Previous studies in vivo reported that processing of primary microRNA (pri-miRNA) is coupled to transcription by RNA polymerase II (RNAP II) and can occur co-transcriptionally. Here we have established a robust in vivo system in which pri-miRNA is transcribed by RNAP II and processed to pre-miRNA in HeLa cell nuclear extracts. We show that both the kinetics and efficiency of pri-miRNA processing are dramatically enhanced in this system compared to that of the corresponding naked pri-miRNA. Moreover, this enhancement is general as it occurs with multiple pri-miRNAs. We also show that nascent pri-miRNA is efficiently processed before it is released from the DNA template. Together, our work directly demonstrates that transcription and pri-miRNA processing are functionally coupled and establishes the first in vivo model systems for this functional coupling and for co-transcriptional processing.


Subject(s)
MicroRNAs/metabolism , RNA Polymerase II/genetics , HeLa Cells , Humans , MicroRNAs/genetics , Nucleic Acid Conformation , RNA Polymerase II/metabolism , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , Transcription, Genetic
15.
Proc Natl Acad Sci U S A ; 112(28): 8608-13, 2015 Jul 14.
Article in English | MEDLINE | ID: mdl-26124092

ABSTRACT

Pre-mRNA splicing is coupled to transcription by RNA polymerase II (RNAP II). We previously showed that U1 small nuclear ribonucleoprotein (snRNP) associates with RNAP II, and both RNAP II and U1 snRNP are also the most abundant factors associated with the protein fused-in-sarcoma (FUS), which is mutated to cause the neurodegenerative disease amyotrophic lateral sclerosis. Here, we show that an antisense morpholino that base-pairs to the 5' end of U1 snRNA blocks splicing in the coupled system and completely disrupts the association between U1 snRNP and both FUS and RNAP II, but has no effect on the association between FUS and RNAP II. Conversely, we found that U1 snRNP does not interact with RNAP II in FUS knockdown extracts. Moreover, using these extracts, we found that FUS must be present during the transcription reaction in order for splicing to occur. Together, our data lead to a model that FUS functions in coupling transcription to splicing via mediating an interaction between RNAP II and U1 snRNP.


Subject(s)
RNA Polymerase II/metabolism , RNA Splicing/physiology , RNA-Binding Protein FUS/physiology , Ribonucleoprotein, U1 Small Nuclear/metabolism , Transcription, Genetic/physiology , Base Sequence , Humans , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/metabolism , RNA-Binding Protein FUS/metabolism
16.
Am J Hum Genet ; 97(2): 302-10, 2015 Aug 06.
Article in English | MEDLINE | ID: mdl-26166480

ABSTRACT

Export of mRNA from the cell nucleus to the cytoplasm is essential for protein synthesis, a process vital to all living eukaryotic cells. mRNA export is highly conserved and ubiquitous. Mutations affecting mRNA and mRNA processing or export factors, which cause aberrant retention of mRNAs in the nucleus, are thus emerging as contributors to an important class of human genetic disorders. Here, we report that variants in THOC2, which encodes a subunit of the highly conserved TREX mRNA-export complex, cause syndromic intellectual disability (ID). Affected individuals presented with variable degrees of ID and commonly observed features included speech delay, elevated BMI, short stature, seizure disorders, gait disturbance, and tremors. X chromosome exome sequencing revealed four missense variants in THOC2 in four families, including family MRX12, first ascertained in 1971. We show that two variants lead to decreased stability of THOC2 and its TREX-complex partners in cells derived from the affected individuals. Protein structural modeling showed that the altered amino acids are located in the RNA-binding domains of two complex THOC2 structures, potentially representing two different intermediate RNA-binding states of THOC2 during RNA transport. Our results show that disturbance of the canonical molecular pathway of mRNA export is compatible with life but results in altered neuronal development with other comorbidities.


Subject(s)
Active Transport, Cell Nucleus/genetics , Chromosomes, Human, X/genetics , Mental Retardation, X-Linked/genetics , Models, Molecular , Mutation, Missense/genetics , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Amino Acid Sequence , Base Sequence , Humans , Mental Retardation, X-Linked/pathology , Molecular Sequence Data , Pedigree , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , Sequence Analysis, DNA , Syndrome
17.
Nucleic Acids Res ; 43(6): 3208-18, 2015 Mar 31.
Article in English | MEDLINE | ID: mdl-25735748

ABSTRACT

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to neurodegeneration remain obscure. We previously found that U1 snRNP is the most abundant FUS interactor. Here, we report that components of the U1 snRNP core particle (Sm proteins and U1 snRNA), but not the mature U1 snRNP-specific proteins (U1-70K, U1A and U1C), co-mislocalize with FUS to the cytoplasm in ALS patient fibroblasts harboring mutations in the FUS nuclear localization signal (NLS). Similar results were obtained in HeLa cells expressing the ALS-causing FUS R495X NLS mutation, and mislocalization of Sm proteins is RRM-dependent. Moreover, as observed with FUS, knockdown of any of the U1 snRNP-specific proteins results in a dramatic loss of SMN-containing Gems. Significantly, knockdown of U1 snRNP in zebrafish results in motor axon truncations, a phenotype also observed with FUS, SMN and TDP-43 knockdowns. Our observations linking U1 snRNP to ALS patient cells with FUS mutations, SMN-containing Gems, and motor neurons indicate that U1 snRNP is a component of a molecular pathway associated with motor neuron disease. Linking an essential canonical splicing factor (U1 snRNP) to this pathway provides strong new evidence that splicing defects may be involved in pathogenesis and that this pathway is a potential therapeutic target.


Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Nuclear Localization Signals/genetics , RNA-Binding Protein FUS/genetics , Ribonucleoprotein, U1 Small Nuclear/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Animals, Genetically Modified , Cytoplasm/metabolism , Gemini of Coiled Bodies/metabolism , Gemini of Coiled Bodies/pathology , Gene Knockdown Techniques , HeLa Cells , Humans , Motor Neurons/metabolism , Motor Neurons/pathology , Mutation , Protein Interaction Domains and Motifs , RNA-Binding Protein FUS/chemistry , RNA-Binding Protein FUS/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/antagonists & inhibitors , Ribonucleoprotein, U1 Small Nuclear/genetics , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , snRNP Core Proteins/genetics , snRNP Core Proteins/metabolism
18.
J Clin Psychopharmacol ; 34(4): 441-5, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24943389

ABSTRACT

OBJECTIVE: Clozapine, an evidence-based treatment of refractory schizophrenia, is associated with increased weight gain and metabolic dysregulation compared with most antipsychotics in short-term clinical trials. However, there are limited data describing comparative long-term metabolic risks. In this report, we examined whether short-term differences persist with long-term exposure to clozapine. METHODS: The data of all patients in a university-based clinic with a psychotic illness or a mood disorder with psychotic features, based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision diagnosis, and treated with an antipsychotic in calendar year 2012 were examined. A total of 307 patients met the criteria; 96 patients were treated with clozapine and the remaining 211 patients were treated with 1 or more non-clozapine antipsychotics. Body mass index, type 2 diabetes, hypertension, dyslipidemia, and obesity were compared. RESULTS: The mean duration of the clozapine treatment was 7.6 years (range, 2 months to 21 y). On all metabolic measures, there were no statistically significant differences between the clozapine and non-clozapine groups (mean body mass index, 31 vs 32; type 2 diabetes, 17% vs 18%; dyslipidemia, 35% vs 38%; hypertension, 32% vs 39%; and obesity, 48% vs 54%). Removing the olanzapine-treated patients (n = 51) from the non-clozapine group did not change the findings. CONCLUSIONS: In this university-based clinic sample with a large number of clozapine-treated patients, we found no evidence of increased risk in any individual measure for those receiving clozapine. Although speculative, the relative contribution of the increased short-term metabolic risk associated with clozapine may be diminished over time because multiple other variables likely also impact metabolic risk during the life span. Although speculative, the relative contribution of the increased short-term metabolic risk associated with clozapine may be diminished over time due to the accumulated impact of other variables that also impact metabolic risk across the life span.


Subject(s)
Antipsychotic Agents/adverse effects , Clozapine/adverse effects , Metabolic Diseases/chemically induced , Metabolic Diseases/metabolism , Psychotic Disorders/drug therapy , Psychotic Disorders/metabolism , Weight Gain/drug effects , Adult , Aged , Cross-Sectional Studies , Diabetes Mellitus, Type 2/chemically induced , Diabetes Mellitus, Type 2/metabolism , Electronic Health Records/standards , Female , Humans , Male , Middle Aged , Obesity/chemically induced , Obesity/metabolism , Schizophrenia/drug therapy , Schizophrenia/metabolism , Weight Gain/physiology , Young Adult
19.
Methods Mol Biol ; 1126: 169-77, 2014.
Article in English | MEDLINE | ID: mdl-24549664

ABSTRACT

Studies over the past several years have revealed that steps in gene expression are extensively coupled to one another both physically and functionally. Recently, in vitro systems were developed for understanding the mechanisms involved in coupling transcription by RNA polymerase II to RNA processing. Here we describe an efficient two-way system for coupling transcription to splicing and a robust three-way system for coupling transcription, splicing, and polyadenylation. In these systems a CMV-DNA construct is incubated in HeLa cell nuclear extracts in the presence of (32)P-UTP to generate the nascent transcript. Transcription is then stopped by addition of α-amanitin followed by continued incubation to allow RNA processing.


Subject(s)
Alternative Splicing/genetics , Molecular Biology/methods , RNA Precursors/genetics , Transcription, Genetic , Cell Extracts/genetics , Cell Extracts/isolation & purification , Cell Nucleus/genetics , HeLa Cells , Humans , Polyadenylation/genetics , RNA Polymerase II/genetics
20.
J Med Genet ; 50(8): 543-51, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23749989

ABSTRACT

BACKGROUND AND AIM: We identified a balanced de novo translocation involving chromosomes Xq25 and 8q24 in an eight year-old girl with a non-progressive form of congenital ataxia, cognitive impairment and cerebellar hypoplasia. METHODS AND RESULTS: Breakpoint definition showed that the promoter of the Protein Tyrosine Kinase 2 (PTK2, also known as Focal Adhesion Kinase, FAK) gene on chromosome 8q24.3 is translocated 2 kb upstream of the THO complex subunit 2 (THOC2) gene on chromosome Xq25. PTK2 is a well-known non-receptor tyrosine kinase whereas THOC2 encodes a component of the evolutionarily conserved multiprotein THO complex, involved in mRNA export from nucleus. The translocation generated a sterile fusion transcript under the control of the PTK2 promoter, affecting expression of both PTK2 and THOC2 genes. PTK2 is involved in cell adhesion and, in neurons, plays a role in axonal guidance, and neurite growth and attraction. However, PTK2 haploinsufficiency alone is unlikely to be associated with human disease. Therefore, we studied the role of THOC2 in the CNS using three models: 1) THOC2 ortholog knockout in C.elegans which produced functional defects in specific sensory neurons; 2) Thoc2 knockdown in primary rat hippocampal neurons which increased neurite extension; 3) Thoc2 knockdown in neuronal stem cells (LC1) which increased their in vitro growth rate without modifying apoptosis levels. CONCLUSION: We suggest that THOC2 can play specific roles in neuronal cells and, possibly in combination with PTK2 reduction, may affect normal neural network formation, leading to cognitive impairment and cerebellar congenital hypoplasia.


Subject(s)
Cerebellum/abnormalities , Chromosomes, Human, Pair 8/genetics , Focal Adhesion Kinase 1/genetics , Nervous System Malformations/genetics , Psychomotor Disorders/genetics , RNA-Binding Proteins/genetics , Translocation, Genetic , Animals , Caenorhabditis elegans/genetics , Cell Line, Transformed , Child , Developmental Disabilities/complications , Developmental Disabilities/genetics , Female , Gene Fusion , Humans , Male , Mice , Mice, Inbred C57BL , Nervous System Malformations/complications , Psychomotor Disorders/complications , Rats
SELECTION OF CITATIONS
SEARCH DETAIL
...