Splicing Reporter Mice Revealed the Evolutionally Conserved Switching Mechanism of Tissue-Specific Alternative Exon Selection

Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. We have been focusing on tissue-specific regulation of mutually exclusive selection of alternative exons because this implies the typical molecular mechanism of alternative splicing regulation and also can be good examples to elicit general rule of “splice code”. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions of how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, we generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, we demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code,” in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in a tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions

Human Umbilical Cord Blood Treatment in a Mouse Model of ALS: Optimization of Cell Dose

Amyotrophic Lateral Sclerosis (ALS) is a multicausal disease characterized by motor neuron degeneration in the spinal cord and brain. Cell therapy may be a promising new treatment for this devastating disorder. We recently showed that a single low dose (10(6) cells) of mononuclear human umbilical cord blood (MNC hUCB) cells administered intravenously to G93A mice delayed symptom progression and modestly prolonged lifespan. The aim of this pre-clinical translation study is to optimize the dose of MNC hUCB cells to retard disease progression in G93A mice. Three different doses of MNC hUCB cells, 10x10(6), 25x10(6) and 50x10(6), were administered intravenously into pre-symptomatic G93A mice. Motor function tests and various assays to determine cell effects were performed on these mice.Our results showed that a cell dose of 25x10(6) cells significantly increased lifespan of mice by 20-25% and delayed disease progression by 15%. The most beneficial effect on decreasing pro-inflammatory cytokines in the brain and spinal cord was found in this group of mice. Human Th2 cytokines were found in plasma of mice receiving 25x10(6) cells, although prevalent human Th1 cytokines were indicated in mice with 50x10(6) cells. High response of splenic cells to mitogen (PHA) was indicated in mice receiving 25x10(6) (mainly) and 10x10(6) cells. Significantly increased lymphocytes and decreased neutrophils in the peripheral blood were found only in animals receiving 25x10(6) cells. Stable reduction in microglia density in both cervical and lumbar spinal cords was also noted in mice administered with 25x10(6) cells.These results demonstrate that treatment for ALS with an appropriate dose of MNC hUCB cells may provide a neuroprotective effect for motor neurons through active involvement of these cells in modulating the host immune inflammatory system response

Mutations in ASXL1 are associated with poor prognosis across the spectrum of malignant myeloid diseases

The ASXL1 gene is one of the most frequently mutated genes in malignant myeloid diseases. The ASXL1 protein belongs to protein complexes involved in the epigenetic regulation of gene expression. ASXL1 mutations are found in myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). They are generally associated with signs of aggressiveness and poor clinical outcome. Because of this, a systematic determination of ASXL1 mutational status in myeloid malignancies should help in prognosis assessment

Internal Ribosomal Entry Site-Mediated Translation Is Important for Rhythmic PERIOD1 Expression

The mouse PERIOD1 (mPER1) plays an important role in the maintenance of circadian rhythm. Translation of mPer1 is directed by both a cap-dependent process and cap-independent translation mediated by an internal ribosomal entry site (IRES) in the 5′ untranslated region (UTR). Here, we compared mPer1 IRES activity with other cellular IRESs. We also found critical region in mPer1 5′UTR for heterogeneous nuclear ribonucleoprotein Q (HNRNPQ) binding. Deletion of HNRNPQ binding region markedly decreased IRES activity and disrupted rhythmicity. A mathematical model also suggests that rhythmic IRES-dependent translation is a key process in mPER1 oscillation. The IRES-mediated translation of mPer1 will help define the post-transcriptional regulation of the core clock genes

CHD1 Remodels Chromatin and Influences Transient DNA Methylation at the Clock Gene frequency

Circadian-regulated gene expression is predominantly controlled by a transcriptional negative feedback loop, and it is evident that chromatin modifications and chromatin remodeling are integral to this process in eukaryotes. We previously determined that multiple ATP–dependent chromatin-remodeling enzymes function at frequency (frq). In this report, we demonstrate that the Neurospora homologue of chd1 is required for normal remodeling of chromatin at frq and is required for normal frq expression and sustained rhythmicity. Surprisingly, our studies of CHD1 also revealed that DNA sequences within the frq promoter are methylated, and deletion of chd1 results in expansion of this methylated domain. DNA methylation of the frq locus is altered in strains bearing mutations in a variety of circadian clock genes, including frq, frh, wc-1, and the gene encoding the frq antisense transcript (qrf). Furthermore, frq methylation depends on the DNA methyltransferase, DIM-2. Phenotypic characterization of Δdim-2 strains revealed an approximate WT period length and a phase advance of approximately 2 hours, indicating that methylation plays only an ancillary role in clock-regulated gene expression. This suggests that DNA methylation, like the antisense transcript, is necessary to establish proper clock phasing but does not control overt rhythmicity. These data demonstrate that the epigenetic state of clock genes is dependent on normal regulation of clock components

Genomic Convergence among ERRα, PROX1, and BMAL1 in the Control of Metabolic Clock Outputs

Metabolic homeostasis and circadian rhythms are closely intertwined biological processes. Nuclear receptors, as sensors of hormonal and nutrient status, are actively implicated in maintaining this physiological relationship. Although the orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) plays a central role in the control of energy metabolism and its expression is known to be cyclic in the liver, its role in temporal control of metabolic networks is unknown. Here we report that ERRα directly regulates all major components of the molecular clock. ERRα-null mice also display deregulated locomotor activity rhythms and circadian period lengths under free-running conditions, as well as altered circulating diurnal bile acid and lipid profiles. In addition, the ERRα-null mice exhibit time-dependent hypoglycemia and hypoinsulinemia, suggesting a role for ERRα in modulating insulin sensitivity and glucose handling during the 24-hour light/dark cycle. We also provide evidence that the newly identified ERRα corepressor PROX1 is implicated in rhythmic control of metabolic outputs. To help uncover the molecular basis of these phenotypes, we performed genome-wide location analyses of binding events by ERRα, PROX1, and BMAL1, an integral component of the molecular clock. These studies revealed the existence of transcriptional regulatory loops among ERRα, PROX1, and BMAL1, as well as extensive overlaps in their target genes, implicating these three factors in the control of clock and metabolic gene networks in the liver. Genomic convergence of ERRα, PROX1, and BMAL1 transcriptional activity thus identified a novel node in the molecular circuitry controlling the daily timing of metabolic processes

The mammalian centrosome and its functional significance

Primarily known for its role as major microtubule organizing center, the centrosome is increasingly being recognized for its functional significance in key cell cycle regulating events. We are now at the beginning of understanding the centrosome’s functional complexities and its major impact on directing complex interactions and signal transduction cascades important for cell cycle regulation. The centrosome orchestrates entry into mitosis, anaphase onset, cytokinesis, G1/S transition, and monitors DNA damage. Recently, the centrosome has also been recognized as major docking station where regulatory complexes accumulate including kinases and phosphatases as well as numerous other cell cycle regulators that utilize the centrosome as platform to coordinate multiple cell cycle-specific functions. Vesicles that are translocated along microtubules to and away from centrosomes may also carry enzymes or substrates that use centrosomes as main docking station. The centrosome’s role in various diseases has been recognized and a wealth of data has been accumulated linking dysfunctional centrosomes to cancer, Alstrom syndrome, various neurological disorders, and others. Centrosome abnormalities and dysfunctions have been associated with several types of infertility. The present review highlights the centrosome’s significant roles in cell cycle events in somatic and reproductive cells and discusses centrosome abnormalities and implications in disease