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1.  ALS-Associated FUS Mutations Result in Compromised FUS Alternative Splicing and Autoregulation 
PLoS Genetics  2013;9(10):e1003895.
The gene encoding a DNA/RNA binding protein FUS/TLS is frequently mutated in amyotrophic lateral sclerosis (ALS). Mutations commonly affect its carboxy-terminal nuclear localization signal, resulting in varying deficiencies of FUS nuclear localization and abnormal cytoplasmic accumulation. Increasing evidence suggests deficiencies in FUS nuclear function may contribute to neuron degeneration. Here we report a novel FUS autoregulatory mechanism and its deficiency in ALS-associated mutants. Using FUS CLIP-seq, we identified significant FUS binding to a highly conserved region of exon 7 and the flanking introns of its own pre-mRNAs. We demonstrated that FUS is a repressor of exon 7 splicing and that the exon 7-skipped splice variant is subject to nonsense-mediated decay (NMD). Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein. Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS. This dynamic regulation of alternative splicing describes a novel mechanism of FUS autoregulation. Given that ALS-associated FUS mutants are deficient in nuclear localization, we examined whether cells expressing these mutants would be deficient in repressing exon 7 splicing. We showed that FUS harbouring R521G, R522G or ΔExon15 mutation (minor, moderate or severe cytoplasmic localization, respectively) directly correlated with respectively increasing deficiencies in both exon 7 repression and autoregulation of its own protein levels. These data suggest that compromised FUS autoregulation can directly exacerbate the pathogenic accumulation of cytoplasmic FUS protein in ALS. We showed that exon 7 skipping can be induced by antisense oligonucleotides targeting its flanking splice sites, indicating the potential to alleviate abnormal cytoplasmic FUS accumulation in ALS. Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration.
Author Summary
FUS/TLS is a frequently mutated gene in amyotrophic lateral sclerosis (ALS). ALS, also known as Lou Gehrig's disease, is characterized by a progressive degeneration of motor neurons. The abnormal cytoplasmic accumulation of mutant FUS protein is a characteristic pathology of ALS; however, recent evidence increasingly suggests deficiencies in FUS nuclear function may also contribute to neurodegeneration in ALS. Here we report a novel autoregulatory mechanism of FUS by alternative splicing and nonsense mediated decay (NMD). We show FUS binds to exon 7 and flanking introns of its own pre-mRNAs. This results in exon skipping, inducing a reading frame shift and subsequent degradation of the splice variants. As such, this mechanism provides a feedback loop that controls the homeostasis of FUS protein levels. This balance is disrupted in ALS-associated FUS mutants, which are deficient in nuclear localization and FUS-dependent alternative splicing. As a result, the abnormal accumulation of mutant FUS protein in ALS neurons goes unchecked and uncontrolled. Our study provides novel insight into the molecular mechanism by which FUS regulates gene expression and new understanding of the role of FUS in disease at the molecular level. This may lead to new potential therapeutic targets for the treatment of ALS.
doi:10.1371/journal.pgen.1003895
PMCID: PMC3814325  PMID: 24204307
2.  The emerging functions of the p53-miRNA network in stem cell biology 
Cell Cycle  2012;11(11):2063-2072.
The p53 pathway plays an essential role in tumor suppression, regulating multiple cellular processes coordinately to maintain genome integrity in both somatic cells and stem cells. Despite decades of research dedicated to p53 function in differentiated somatic cells, we are just starting to understand the complexity of the p53 pathway in the biology of pluripotent stem cells and tissue stem cells. Recent studies have demonstrated that p53 suppresses proliferation, promotes differentiation of embryonic stem (ES) cells and constitutes an important barrier to somatic reprogramming. In addition, emerging evidence reveals the role of the p53 network in the self-renewal, proliferation and genomic integrity of adult stem cells. Interestingly, non-coding RNAs, and microRNAs in particular, are integral components of the p53 network, regulating multiple p53-controlled biological processes to modulate the self-renewal and differentiation potential of a variety of stem cells. Thus, elucidation of the p53-miRNA axis in stem cell biology may generate profound insights into the mechanistic overlap between malignant transformation and stem cell biology.
doi:10.4161/cc.20207
PMCID: PMC3368858  PMID: 22580472
embryonic stem cells; induced pluripotent stem cells; microRNA; miR-34; p53; stem cells
3.  Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs 
Nature neuroscience  2012;15(11):1488-1497.
FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate–containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.
doi:10.1038/nn.3230
PMCID: PMC3586380  PMID: 23023293
4.  miR-34 miRNAs provide a barrier for somatic cell reprogramming 
Nature cell biology  2011;13(11):1353-1360.
Somatic reprogramming induced by defined transcription factors is a low efficiency process that is enhanced by p53 deficiency 1-5. To date, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation 1, 3, suggesting additional p53 targets may regulate this process. Here, we demonstrated that mir-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. mir-34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of mir-34a promoted iPSC generation without compromising self-renewal and differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three mir-34 miRNAs acted cooperatively in this process. Taken together, our findings identified mir-34 miRNAs as novel p53 targets that play an essential role in restraining somatic reprogramming.
doi:10.1038/ncb2366
PMCID: PMC3541684  PMID: 22020437
5.  Activation of cryptic 3′ splice sites within introns of cellular genes following gene entrapment 
Nucleic Acids Research  2004;32(9):2912-2924.
Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3′-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3′ splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3′ splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3′ splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3′ splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3′ splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3′ splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3′ processing and polyadenylation of cellular transcripts.
doi:10.1093/nar/gkh604
PMCID: PMC419606  PMID: 15155860
6.  A generalizable pre-clinical research approach for orphan disease therapy 
With the advent of next-generation DNA sequencing, the pace of inherited orphan disease gene identification has increased dramatically, a situation that will continue for at least the next several years. At present, the numbers of such identified disease genes significantly outstrips the number of laboratories available to investigate a given disorder, an asymmetry that will only increase over time. The hope for any genetic disorder is, where possible and in addition to accurate diagnostic test formulation, the development of therapeutic approaches. To this end, we propose here the development of a strategic toolbox and preclinical research pathway for inherited orphan disease. Taking much of what has been learned from rare genetic disease research over the past two decades, we propose generalizable methods utilizing transcriptomic, system-wide chemical biology datasets combined with chemical informatics and, where possible, repurposing of FDA approved drugs for pre-clinical orphan disease therapies. It is hoped that this approach may be of utility for the broader orphan disease research community and provide funding organizations and patient advocacy groups with suggestions for the optimal path forward. In addition to enabling academic pre-clinical research, strategies such as this may also aid in seeding startup companies, as well as further engaging the pharmaceutical industry in the treatment of rare genetic disease.
doi:10.1186/1750-1172-7-39
PMCID: PMC3458970  PMID: 22704758
Orphan disease therapy; Preclinical drug development; Generalizable screening methods; Translational toolbox
7.  Identification and characterization of Dlc1 isoforms in the mouse and study of the biological function of a single gene trapped isoform 
BMC Biology  2010;8:17.
Background
The Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied.
Results
In addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1gt/gt embryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an in vitro scratch assay.
Conclusions
The mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.
doi:10.1186/1741-7007-8-17
PMCID: PMC2839985  PMID: 20199662
8.  The International Gene Trap Consortium Website: a portal to all publicly available gene trap cell lines in mouse 
Nucleic Acids Research  2005;34(Database issue):D642-D648.
Gene trapping is a method of generating murine embryonic stem (ES) cell lines containing insertional mutations in known and novel genes. A number of international groups have used this approach to create sizeable public cell line repositories available to the scientific community for the generation of mutant mouse strains. The major gene trapping groups worldwide have recently joined together to centralize access to all publicly available gene trap lines by developing a user-oriented Website for the International Gene Trap Consortium (IGTC). This collaboration provides an impressive public informatics resource comprising ∼45 000 well-characterized ES cell lines which currently represent ∼40% of known mouse genes, all freely available for the creation of knockout mice on a non-collaborative basis. To standardize annotation and provide high confidence data for gene trap lines, a rigorous identification and annotation pipeline has been developed combining genomic localization and transcript alignment of gene trap sequence tags to identify trapped loci. This information is stored in a new bioinformatics database accessible through the IGTC Website interface. The IGTC Website () allows users to browse and search the database for trapped genes, BLAST sequences against gene trap sequence tags, and view trapped genes within biological pathways. In addition, IGTC data have been integrated into major genome browsers and bioinformatics sites to provide users with outside portals for viewing this data. The development of the IGTC Website marks a major advance by providing the research community with the data and tools necessary to effectively use public gene trap resources for the large-scale characterization of mammalian gene function.
doi:10.1093/nar/gkj097
PMCID: PMC1347459  PMID: 16381950
9.  The High-Mobility-Group Box Protein SSRP1/T160 Is Essential for Cell Viability in Day 3.5 Mouse Embryos 
Molecular and Cellular Biology  2003;23(15):5301-5307.
The high-mobility-group (HMG) SSRP1 protein is a member of a conserved chromatin-remodeling complex (FACT/DUF/CP) implicated in DNA replication, basal and regulated transcription, and DNA repair. To assist in the functional analysis of SSRP1, the Ssrp1 gene was targeted in murine embryonic stem cells, and the mutation was introduced into the germ line. Embryos homozygous for the targeted allele die soon after implantation, and preimplantation blastocysts are defective for cell outgrowth and/or survival in vitro. The Ssrp1 mutation was also crossed into a p53 null background without affecting growth and/or survival defects caused by loss of Ssrp1 function. Thus, Ssrp1 appears to encode nonredundant and p53-independent functions that are essential for cell viability.
doi:10.1128/MCB.23.15.5301-5307.2003
PMCID: PMC165710  PMID: 12861016

Results 1-9 (9)