Causal ALS genes impact the MHC class II antigen presentation pathway.

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.

A U2-snRNP-independent role of SF3b in promoting mRNA export.

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.

The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex.

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.

The anti-tumor drug E7107 reveals an essential role for SF3b in remodeling U2 snRNP to expose the branch point-binding region.

Duplex formation between the branch point-binding region (BBR) of U2 snRNA and the branch point sequence (BPS) in the intron is essential for splicing. Both the BBR and BPS interact with the U2 small nuclear ribonucleoprotein (snRNP)-associated SF3b complex, which is the target of the anti-tumor drug E7107. We show that E7107 blocks spliceosome assembly by preventing tight binding of U2 snRNP to pre-mRNA. E7107 has no apparent effect on U2 snRNP integrity. Instead, E7107 abolishes an ATP-dependent conformational change in U2 snRNP that exposes the BBR. We conclude that SF3b is required for this remodeling, which exposes the BBR for tight U2 snRNP binding to pre-mRNA.

THOC2 Mutations Implicate mRNA-Export Pathway in X-Linked Intellectual Disability.

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.

FUS functions in coupling transcription to splicing by mediating an interaction between RNAP II and U1 snRNP.

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.

ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex.

The conserved TREX mRNA export complex is known to contain UAP56, Aly, Tex1, and the THO complex. Here, we carried out proteomic analysis of immunopurified human TREX complex and identified the protein CIP29 as the only new component with a clear yeast relative (known as Tho1). Tho1 is known to function in mRNA export, and we provide evidence that CIP29 likewise functions in this process. Like the known TREX components, a portion of CIP29 localizes in nuclear speckle domains, and its efficient recruitment to mRNA is both splicing- and cap-dependent. We show that UAP56 mediates an ATP-dependent interaction between the THO complex and both CIP29 and Aly, indicating that TREX assembly is ATP-dependent. Using recombinant proteins expressed in Escherichia coli, we show that UAP56, Aly, and CIP29 form an ATP-dependent trimeric complex, and UAP56 bridges the interaction between CIP29 and Aly. We conclude that the interaction of two conserved export proteins, CIP29 and Aly, with UAP56 is strictly regulated by ATP during assembly of the TREX complex.

U1 snRNP is mislocalized in ALS patient fibroblasts bearing NLS mutations in FUS and is required for motor neuron outgrowth in zebrafish.

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.

Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export.

We previously showed that mRNAs synthesized from three genes that naturally lack introns contain a portion of their coding sequence, known as a cytoplasmic accumulation region (CAR), which is essential for stable accumulation of the intronless mRNAs in the cytoplasm. The CAR in each mRNA is unexpectedly large, ranging in size from ∼160 to 285 nt. Here, we identified one or more copies of a 10-nt consensus sequence in each CAR. To determine whether this element (designated CAR-E) functions in cytoplasmic accumulation of intronless mRNA, we multimerized the most conserved CAR-E and inserted it upstream of ß-globin cDNA, which is normally retained/degraded in the nucleus. Significantly, the tandem CAR-E, but not its antisense counterpart, rescued cytoplasmic accumulation of ß-globin cDNA transcripts. Moreover, dinucleotide mutations in the CAR-E abolished this rescue. We show that the CAR-E, but not the mutant CAR-E, associates with components of the TREX mRNA export machinery, the Prp19 complex and U2AF2. Moreover, knockdown of these factors results in nuclear retention of the intronless mRNAs. Together, these data suggest that the CAR-E promotes export of intronless mRNA by sequence-dependent recruitment of the mRNA export machinery.

SF3B1 and other novel cancer genes in chronic lymphocytic leukemia.

BACKGROUND: The somatic genetic basis of chronic lymphocytic leukemia, a common and clinically heterogeneous leukemia occurring in adults, remains poorly understood. METHODS: We obtained DNA samples from leukemia cells in 91 patients with chronic lymphocytic leukemia and performed massively parallel sequencing of 88 whole exomes and whole genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in this disease. RESULTS: Nine genes that are mutated at significant frequencies were identified, including four with established roles in chronic lymphocytic leukemia (TP53 in 15% of patients, ATM in 9%, MYD88 in 10%, and NOTCH1 in 4%) and five with unestablished roles (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X). SF3B1, which functions at the catalytic core of the spliceosome, was the second most frequently mutated gene (with mutations occurring in 15% of patients). SF3B1 mutations occurred primarily in tumors with deletions in chromosome 11q, which are associated with a poor prognosis in patients with chronic lymphocytic leukemia. We further discovered that tumor samples with mutations in SF3B1 had alterations in pre-messenger RNA (mRNA) splicing. CONCLUSIONS: Our study defines the landscape of somatic mutations in chronic lymphocytic leukemia and highlights pre-mRNA splicing as a critical cellular process contributing to chronic lymphocytic leukemia.

A naturalistic comparison of the long-term metabolic adverse effects of clozapine versus other antipsychotics for patients with psychotic illnesses.

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.

A de novo X;8 translocation creates a PTK2-THOC2 gene fusion with THOC2 expression knockdown in a patient with psychomotor retardation and congenital cerebellar hypoplasia.

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.

Export and stability of naturally intronless mRNAs require specific coding region sequences and the TREX mRNA export complex.

A great deal is known about the export of spliced mRNAs, but little is known about the export of mRNAs encoded by human cellular genes that naturally lack introns. Here, we investigated the requirements for export of three naturally intronless mRNAs (HSPB3, IFN-α1, and IFN-ß1). Significantly, we found that all three mRNAs are stable and accumulate in the cytoplasm, whereas size-matched random RNAs are unstable and detected only in the nucleus. A portion of the coding region confers this stability and cytoplasmic localization on the naturally intronless mRNAs and a cDNA transcript, which is normally retained in the nucleus and degraded. A polyadenylation signal, TREX mRNA export components, and the mRNA export receptor TAP are required for accumulation of the naturally intronless mRNAs in the cytoplasm. We conclude that naturally intronless mRNAs contain specific sequences that result in efficient packaging into the TREX mRNA export complex, thereby supplanting the splicing requirement for efficient mRNA export.

TREX, SR proteins and export of mRNA.

The machineries involved in gene expression are highly conserved from yeast to metazoans. However, a fundamental difference between these organisms is that most yeast genes lack introns whereas the converse is true in higher organisms. Recent studies of the TREX complex, which functions in mRNA export, unexpectedly revealed that this complex is recruited by the transcription machinery in yeast whereas the TREX complex appears to be recruited by the splicing machinery in mammals. Studies during the past year also revealed a possible conserved role for SR protein dephosphorylation in regulating the interaction between SR proteins and the mRNA export receptor TAP (Mex67 in yeast). There is also an interesting possibility that an SR protein-TREX complex interaction is a conserved part of the mRNA export machinery.

A model in vitro system for co-transcriptional splicing.

A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous small exons surrounded by large introns. Abundant evidence indicates that splicing to excise introns occurs co-transcriptionally, prior to release of the nascent transcript from RNAP II. Here, we established an efficient model system for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs immobilized on beads generate RNAP II transcripts containing two exons and an intron. Consistent with previous work, our data indicate that elongating nascent transcripts are tethered to RNAP II on the immobilized DNA template. We show that nascent transcripts that reach full length, but are still attached to RNAP II, are efficiently spliced. When the nascent transcript is cleaved within the intron using RNase H, both the 5' and 3' cleavage fragments are detected in the bound fraction, where they undergo splicing. Together, our work establishes a system for co-transcriptional splicing in vitro, in which the spliceosome containing the 5' and 3' exons are tethered to RNAP II for splicing.

Coupling transcription, splicing and mRNA export.

Previous studies have led to the view that mRNAs are transported from the nucleus to the cytoplasm by machinery that is conserved from yeast to humans. Moreover, this machinery is coupled both physically and functionally to the pre-mRNA splicing machinery. During the past year, important new insights into the mechanisms behind this coupling have been made. In addition, recent advances have revealed mechanisms for the co-transcriptional loading of the export machinery on to mRNAs. Finally, a newly identified link between the nuclear exosome and the machineries required for transcription, 3'-end formation and mRNA export suggests that proper mRNP formation is co-transcriptionally monitored.

Splicing promotes rapid and efficient mRNA export in mammalian cells.

The numerous steps in protein gene expression are extensively coupled to one another through complex networks of physical and functional interactions. Indeed, >25 coupled reactions, often reciprocal, have been documented among such steps as transcription, capping, splicing, and polyadenylation. Coupling is usually not essential for gene expression, but instead enhances the rate and/or efficiency of reactions and, physiologically, may serve to increase the fidelity of gene expression. Despite numerous examples of coupling in gene expression, whether splicing enhances mRNA export still remains controversial. Although splicing was originally reported to promote export in both mammalian cells and Xenopus oocytes, it was subsequently concluded that this was not the case. These newer conclusions were surprising in light of the observations that the mRNA export machinery colocalizes with splicing factors in the nucleus and that splicing promotes recruitment of the export machinery to mRNA. We therefore reexamined the relationship between splicing and mRNA export in mammalian cells by using FISH, in combination with either transfection or nuclear microinjection of plasmid DNA. Together, these analyses indicate that both the kinetics and efficiency of mRNA export are enhanced 6- to 10-fold (depending on the construct) for spliced mRNAs relative to their cDNA counterparts. We conclude that splicing promotes mRNA export in mammalian cells and that the functional coupling between splicing and mRNA export is a conserved and general feature of gene expression in higher eukaryotes.

Human DDX3 functions in translation and interacts with the translation initiation factor eIF3.

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3.

Development of NAMASTE (New Anxiety Management Algorithm Standardizing Treatment Experience) and Implementation in Primary Care.

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.

A Murine Model of Chronic Lymphocytic Leukemia Based on B Cell-Restricted Expression of Sf3b1 Mutation and Atm Deletion.

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.