CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion.

CRISPR is widely used to disrupt gene function by inducing small insertions and deletions. Here, we show that some single-guide RNAs (sgRNAs) can induce exon skipping or large genomic deletions that delete exons. For example, CRISPR-mediated editing of ß-catenin exon 3, which encodes an autoinhibitory domain, induces partial skipping of the in-frame exon and nuclear accumulation of ß-catenin. A single sgRNA can induce small insertions or deletions that partially alter splicing or unexpected larger deletions that remove exons. Exon skipping adds to the unexpected outcomes that must be accounted for, and perhaps taken advantage of, in CRISPR experiments.

NEAT1 is Required for Survival of Breast Cancer Cells Through FUS and miR-548.

Increasing evidence shows that long noncoding RNAs (lncRNAs) have important roles in the regulation of multiple cellular processes, including cell division, cell growth, and apoptosis, as well as cancer metastasis and neurological disease progression; however, the mechanism of how lncRNAs regulate these processes is not well established. In this study, we demonstrated that downregulating the expression of the lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in breast cancer cells inhibited cell growth and induced cell apoptosis. In addition, the RNA-binding protein fused in sarcoma/translocated in liposarcoma (FUS/TLS) physically interacted with NEAT1, and reducing the expression of FUS/TLS also induced cell apoptosis. Multiple miRNAs were identified as regulators of NEAT1, but only overexpression of miR-548ar was able to decrease NEAT1 expression and promote apoptosis. These results indicate a novel interaction between NEAT1, miR-548ar-3p, and FUS and their role in the regulation of breast cancer cell apoptosis.

Partial loss of TDP-43 function causes phenotypes of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease that causes motor neuron degeneration, progressive motor dysfunction, paralysis, and death. Although multiple causes have been identified for this disease, >95% of ALS cases show aggregation of transactive response DNA binding protein (TDP-43) accompanied by its nuclear depletion. Therefore, the TDP-43 pathology may be a converging point in the pathogenesis that originates from various initial triggers. The aggregation is thought to result from TDP-43 misfolding, which could generate cellular toxicity. However, the aggregation as well as the nuclear depletion could also lead to a partial loss of TDP-43 function or TDP-43 dysfunction. To investigate the impact of TDP-43 dysfunction, we generated a transgenic mouse model for a partial loss of TDP-43 function using transgenic RNAi. These mice show ubiquitous transgene expression and TDP-43 knockdown in both the periphery and the central nervous system (CNS). Strikingly, these mice develop progressive neurodegeneration prominently in cortical layer V and spinal ventral horn, motor dysfunction, paralysis, and death. Furthermore, examination of splicing patterns of TDP-43 target genes in human ALS revealed changes consistent with TDP-43 dysfunction. These results suggest that the CNS, particularly motor neurons, possess a heightened vulnerability to TDP-43 dysfunction. Additionally, because TDP-43 knockdown predominantly occur in astrocytes in the spinal cord of these mice, our results suggest that TDP-43 dysfunction in astrocytes is an important driver for motor neuron degeneration and clinical phenotypes of ALS.

Staufen1 senses overall transcript secondary structure to regulate translation.

Human Staufen1 (Stau1) is a double-stranded RNA (dsRNA)-binding protein implicated in multiple post-transcriptional gene-regulatory processes. Here we combined RNA immunoprecipitation in tandem (RIPiT) with RNase footprinting, formaldehyde cross-linking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1-binding sites transcriptome wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3' untranslated regions (UTRs) or in 'strongly distal' 3' UTRs. Stau1 also interacts with actively translating ribosomes and with mRNA coding sequences (CDSs) and 3' UTRs in proportion to their GC content and propensity to form internal secondary structure. On mRNAs with high CDS GC content, higher Stau1 levels lead to greater ribosome densities, thus suggesting a general role for Stau1 in modulating translation elongation through structured CDS regions. Our results also indicate that Stau1 regulates translation of transcription-regulatory proteins.

Downregulation of microRNA-9 in iPSC-derived neurons of FTD/ALS patients with TDP-43 mutations.

Transactive response DNA-binding protein 43 (TDP-43) is a major pathological protein in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). There are many disease-associated mutations in TDP-43, and several cellular and animal models with ectopic overexpression of mutant TDP-43 have been established. Here we sought to study altered molecular events in FTD and ALS by using induced pluripotent stem cell (iPSC) derived patient neurons. We generated multiple iPSC lines from an FTD/ALS patient with the TARDBP A90V mutation and from an unaffected family member who lacked the mutation. After extensive characterization, two to three iPSC lines from each subject were selected, differentiated into postmitotic neurons, and screened for relevant cell-autonomous phenotypes. Patient-derived neurons were more sensitive than control neurons to 100 nM straurosporine but not to other inducers of cellular stress. Three disease-relevant cellular phenotypes were revealed under staurosporine-induced stress. First, TDP-43 was localized in the cytoplasm of a higher percentage of patient neurons than control neurons. Second, the total TDP-43 level was lower in patient neurons with the A90V mutation. Third, the levels of microRNA-9 (miR-9) and its precursor pri-miR-9-2 decreased in patient neurons but not in control neurons. The latter is likely because of reduced TDP-43, as shRNA-mediated TDP-43 knockdown in rodent primary neurons also decreased the pri-miR-9-2 level. The reduction in miR-9 expression was confirmed in human neurons derived from iPSC lines containing the more pathogenic TARDBP M337V mutation, suggesting miR-9 downregulation might be a common pathogenic event in FTD/ALS. These results show that iPSC models of FTD/ALS are useful for revealing stress-dependent cellular defects of human patient neurons containing rare TDP-43 mutations in their native genetic contexts.

An atypical HLH protein OsLF in rice regulates flowering time and interacts with OsPIL13 and OsPIL15.

In plants, flowering as a crucial developmental event is highly regulated by both genetic programs and environmental signals. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flowering induction. In this study, we isolated the OsLF gene by its association with the T-DNA insertion in the rice late flowering mutant named A654. The OsLF gene encodes an atypical HLH protein composed of 419 amino acids (aa). Overexpression of the OsLF gene in wild type rice recapitulated the late flowering phenotype of A654, indicating that the OsLF gene negatively regulates flowering. Flowering genes downstream of OsPRR1 such as OsGI and Hd1 were down regulated in the A654 mutant. Yeast two hybrid and colocalization assays revealed that OsLF interacts strongly with OsPIL13 and OsPIL15 that are potentially involved in light signaling. In addition, OsPIL13 and OsPIL15 colocalize with OsPRR1, an ortholog of the Arabidopsis APRR1 gene that controls photoperiodic flowering response through clock function. Together, these results suggest that overexpression of OsLF might repress expression of OsGI and Hd1 by competing with OsPRR1 in interacting with OsPIL13 and OsPIL15 and thus induce late flowering.

Overexpression of transcription factor OsLFL1 delays flowering time in Oryza sativa.

Flowering time is regulated by genetic programs and environment signals in plants. Genetic analysis of flowering time mutants is instrumental in dissecting the regulatory pathways of flower induction. Genotype W378 is a rice (Oryza sativa) late-flowering mutant selected from our collections of T-DNA insertion line. The T-DNA flanking gene in mutant W378 codes OsLFL1 (O. sativa LEC2 and FUSCA3 Like 1), a putative B3 DNA-binding domain-containing transcription factor. In wild-type rice OsLFL1 is expressed exclusively in spikes and young embryos, while in mutant W378 it is ectopically expressed. Introduction of OsLFL1-RNAi into mutant W378 successfully down-regulated OsLFL1 expression and restored flowering to almost normal time, indicating that overexpression of OsLFL1 confers late flowering for mutant W378. The flowering-promoting gene Ehd1 and its downstream genes are all down-regulated in W378. Thus, overexpression of OsLFL1 might delay the flowering of W378 by repressing the expression of Ehd1.

Ectopic expression of OsLFL1 in rice represses Ehd1 by binding on its promoter.

B3 domain was identified as a novel DNA-binding motif specific to higher plant species. The B3 proteins play important roles in plant development. In the mutant W378, the mutant gene coding OsLFL1, a putative B3 transcription factor gene, was ectopically expressed. In this study, it was found that the flowering promoting gene Ehd1 and its putative downstream genes were all repressed by OsLFL1. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) analyses suggest that OsLFL1 binds to the RY cis-elements (CATGCATG) in the promoter of the Ehd1 gene. Thus, ectopically expressed OsLFL1 might repress Ehd1 via binding directly to the RY cis-elements in its promoter.

[Structure and expression analysis of the gama-glutamylcysteine synthetase gene in rice].

Gama-glutamylcysteine synthetase (GCS) is a rate-limiting enzyme in GSH biosynthesis. The GCS gene has been cloned in Arabidopsis thaliana and other plants, but has still not been reported in rice. From rice mutant population generated from T-DNA insertion, we cloned the rice GCS gene from mutant L395 by T-DNA tag cloning method, and named it OsGCS (Genbank accession No. AJ508915). Full length OsGCS cDNA clones were obtained from a rice cDNA library by the PCR method. A comparison of the genome and cDNA sequence (Genbank accession No. AJ508916) shows that OsGCS gene is composed of 15 exons and 14 introns and coding a 493-amino acid protein. The OsGCS gene is highly homologous with the AtGCS gene in the coding region but completely different in the promoter region. The putative transcription start site (TSS) confirmed by RT-PCR was located 211 bp upstream of the translation start codon "ATG". In mutant L395, a single T-DNA copy was integrated between the second intron and second exon of OsGCS gene, causing one nucleotide deletion in the second exon and two nucleotide deletions in the second intron. No significant differences were found in Cd(2+) stress tolerance, rice GCS gene expression level and GSH content between mutant L395 and Zhonghua 11. It is possible that another GCS gene on chromosome 7 might complement function of OsGCS gene on chromosome 5.