Summary The RNA-binding proteins Rbfox1/2/3 regulate alternative splicing in the nervous system, and disruption of Rbfox1 has been implicated in autism. However, comprehensive identification of functional Rbfox targets has been challenging. Here we performed HITS-CLIP for all three Rbfox family members to globally map, at a single-nucleotide resolution, their in vivo RNA interaction sites in the mouse brain. We found that the two guanines in the Rbfox-binding motif UGCAUG are critical for protein-RNA interactions and crosslinking. Using integrative modeling, these interaction sites combined with additional datasets defined 1,059 direct Rbfox target alternative splicing events. Over half of the quantifiable targets show dynamic changes during brain development. Of particular interest are 111 events from 48 candidate autism-susceptibility genes, including syndromic autism genes Shank3, Cacna1c, and Tsc2. Alteration of Rbfox targets in some autistic brains is correlated with down-regulation of all three Rbfox proteins, supporting the potential clinical relevance of the splicing- regulatory network.
The precise regulation of many alternative splicing (AS) events by specific splicing factors is essential to determine tissue types and developmental stages. However, the molecular basis of tissue-specific AS regulation and the properties of splicing regulatory networks (SRNs) are poorly understood. Here we comprehensively predict the targets of the brain-and muscle-specific splicing factor Fox-1 (A2BP1) and its paralog Fox-2 (RBM9) and systematically define the corresponding SRNs genome-wide. Fox-1/2 are conserved from worm to human, and specifically recognize the RNA element UGCAUG. We integrate Fox-1/2-binding specificity with phylogenetic conservation, splicing microarray data, and additional computational and experimental characterization. We predict thousands of Fox-1/2 targets with conserved binding sites, at a false discovery rate (FDR) of ∼24%, including many validated experimentally, suggesting a surprisingly extensive SRN. The preferred position of the binding sites differs according to AS pattern, and determines either activation or repression of exon recognition by Fox-1/2. Many predicted targets are important for neuromuscular functions, and have been implicated in several genetic diseases. We also identified instances of binding site creation or loss in different vertebrate lineages and human populations, which likely reflect fine-tuning of gene expression regulation during evolution.[Keywords: Tissue-specific alternative splicing; splicing regulatory network; Fox-1/A2BP1; Fox-2/RBM9; UGCAUG; comparative genomics] Supplemental material is available at http://www.genesdev.org.Received June 6, 1007; revised version accepted July 28, 2008.The sequencing of complete genomes revealed that complex metazoans, including mammals, have only slightly more genes than unicellular yeast (International Human Genome Sequencing Consortium 2001). Organismal complexity must have resulted largely from mechanisms for diversifying the expression products, and the temporal and spatial patterns, from a limited set of genes. It is crucial to understand how gene expression is orchestrated to determine developmental stages, specify cell types, and respond to external stimuli (Maniatis and Reed 2002). Alternative splicing (AS), the process for removing introns from pre-mRNA transcripts and joining exons in different combinations, is an essential step of post-transcriptional regulation (Cartegni et al. 2002;Black 2003). In mammals, more than two-thirds of genes are alternatively spliced (Johnson et al. 2003). The choice of exons and splice sites is largely determined by many RNA-binding proteins, or splicing factors, which interact with cis-regulatory elements to activate or repress particular splicing events.Many splicing factors have restricted and dynamic expression patterns, and play important roles in tissue-specific or developmentally regulated splicing of particular transcripts. However, the mechanisms and impact of these AS events remain poorly understood. A well-studied example is Sxl, Tra, Tra-2, and several other s...
Key Points Adult patients with ETP-ALL/LBL have poor long-term outcomes. Novel therapies are urgently needed for adult patients with ETP-ALL/LBL.
We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3’ of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.
How do cells discriminate between selectively edited mRNAs that encode new protein isoforms, and dsRNA-induced, promiscuously edited RNAs that encode nonfunctional, mutant proteins? We have developed a Xenopus oocyte model system which shows that a variety of hyperedited, inosine-containing RNAs are specifically retained in the nucleus. To uncover the mechanism of inosine-induced retention, HeLa cell nuclear extracts were used to isolate a multiprotein complex that binds specifically and cooperatively to inosine-containing RNAs. This complex contains the inosine-specific RNA binding protein p54(nrb), the splicing factor PSF, and the inner nuclear matrix structural protein matrin 3. We provide evidence that one function of the complex identified here is to anchor hyperedited RNAs to the nuclear matrix, while allowing selectively edited mRNAs to be exported.
SF2/ASF is a prototypical SR protein, with important roles in splicing and other aspects of mRNA metabolism. SFRS1 (SF2/ASF) is a potent proto-oncogene with abnormal expression in many tumors. We found that SF2/ASF negatively autoregulates its expression to maintain homeostatic levels. We characterized six SF2/ASF alternatively spliced mRNA isoforms: the major isoform encodes full-length protein, whereas the others are either retained in the nucleus or degraded by NMD. Unproductive splicing accounts for only part of the autoregulation, which occurs primarily at the translational level. The effect is specific to SF2/ASF and requires RRM2. The ultraconserved 3′UTR is necessary and sufficient for downregulation. SF2/ASF overexpression shifts the distribution of target mRNA towards mono-ribosomes, and translational repression is partly independent of Dicer and a 5′ cap. Thus, multiple post-transcriptional and translational mechanisms are involved in fine-tuning the expression of SF2/ASF.
Mutations in PLA2G6 (PARK14) cause neurodegenerative disorders in humans, including autosomal recessive neuroaxonal dystrophy and early-onset parkinsonism. We show that loss of iPLA2-VIA, the fly homolog of PLA2G6, reduces lifespan, impairs synaptic transmission, and causes neurodegeneration. Phospholipases typically hydrolyze glycerol phospholipids, but loss of iPLA2-VIA does not affect the phospholipid composition of brain tissue but rather causes an elevation in ceramides. Reducing ceramides with drugs, including myriocin or desipramine, alleviates lysosomal stress and suppresses neurodegeneration. iPLA2-VIA binds the retromer subunits Vps35 and Vps26 and enhances retromer function to promote protein and lipid recycling. Loss of iPLA2-VIA impairs retromer function, leading to a progressive increase in ceramide. This induces a positive feedback loop that affects membrane fluidity and impairs retromer function and neuronal function. Similar defects are observed upon loss of vps26 or vps35 or overexpression of α-synuclein, indicating that these defects may be common in Parkinson disease.
BackgroundPlasmablastic lymphoma (PBL) is a rare aggressive neoplasm with lymphoid and plasmacytic differentiation that is commonly associated with immunodeficiency and an unfavorable prognosis. Clinicopathologic features have been largely derived from cases reports and small series with limited outcome analyses.Patients and methodsThe demographic, clinicopathologic features, and clinical outcomes of a cohort of 61 patients with PBL were reviewed and analyzed.ResultsPatients had a median age of 49 years (range 21–83 years) and most (49/61; 80 %) were men. Human immunodeficiency virus (HIV) status was available for 50 patients: 20 were HIV-positive and 30 HIV-negative. Twenty-three patients were immunocompetent. Abdominal/gastrointestinal complaints were the most common presenting symptoms, reported in 14 of 47 (30 %) of patients. At presentation, 24 of 43 (56 %) patients had stage III or IV disease. Epstein-Barr virus (EBV) was detected in 40 of 57 (70 %) cases. MYC rearrangement was identified in 10/15 (67 %) cases assessed, and MYC overexpression was seen in all cases assessed regardless of MYC rearrangement status. HIV-positive patients were significantly younger than those who were HIV-negative (median 42 vs. 58 years; p = 0.006). HIV-positive patients were also significantly more likely to have EBV-positive disease compared with HIV-negative patients (19/19, 100 % vs. 15/29, 52 %; p = 0.002). Patients who received CHOP chemotherapy tended to have better overall survival (OS) compared with those who received hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyper-CVAD) (p = 0.078). HIV status had no impact on OS. Patients with EBV-positive PBL had a better event-free survival (EFS) (p = 0.047) but not OS (p = 0.306). Notably, OS was adversely impacted by age ≥50 years (p = 0.013), stage III or IV disease (p = <0.001), and lymph node involvement (p = 0.008).ConclusionsThe most significant prognostic parameters in patients with PBL are age, stage, and, to a lesser extent, EBV status. In this study, two-thirds of PBL cases assessed were associated with MYC rearrangement and all showed MYC overexpression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0163-z) contains supplementary material, which is available to authorized users.
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