Biallelic variants in RBM42 cause a multisystem disorder with neurological,facial,cardiac,and musculoskeletal involvement

Here,we report a previously unrecognized syndromic neurodevelopmental disorder associated with biallelic loss-of-function variants in the RBM42 gene.The patient is a 2-year-old female with severe central nervous system(CNs)abnormalities,hypotonia,hearing loss,congenital heart defects,and dysmorphic facial features.Familial whole-exome sequencing(WEs)reveals that the patient has two compound heterozygous variants,c.304C>T(p.R102*)and c.1312G>A(p.A438T),in the RBM42 gene which encodes an integral component of splicing complex in the RNA-binding motif protein family.The p.A438T variant is in the RRM domain which impairs RBM42 pro-tein stability in vivo.Additionally,p.A438T disrupts the interaction of RBM42 with hnRNP K,which is the causa-tive gene for Au-Kline syndrome with overlapping disease characteristics seen in the index patient.The human R102*or A438T mutant protein failed to fully rescue the growth defects of RBM42 ortholog knockout△FgRbp1 in Fusarium while it was rescued by the wild-type(WT)human RBM42.A mouse model carying Rbm42 compound heterozygous variants,c.280C>T(p.Q94*)and c.1306_1308delinsACA(p.A436T),demonstrated gross fetal develop-mental defects and most of the double mutant animals died by E13.5.RNA-seq data confirmed that Rbm42 was involved in neurological and myocardial functions with an essential role in alternative splicing(As).Overall,we present clinical,genetic,and functional data to demonstrate that defects in RBM42 constitute the underlying etiology of a new neurodevelopmental disease which links the dysregulation of global AS to abnormal embryonic development.

Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/ Cas9-mediated knock-in

In mammalian cells,long noncoding RNAs(lncRNAs)form complexes with proteins to execute various biological functions such as gene transcription,RNA processing and other signaling activities.However,methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited.Here,we report the development of CERTIS(CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System)to visualize and isolate endogenous lncRNA,by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR7Cas9 technology.In this study,we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1.In addition,CERTIS displayed superior performance on both short-and long-term tracking of NEAT1 dynamics in live cells.We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors.Moreover,RNA Immunoprecipitation(RIP)of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle.Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.

Correction of β-thalassemia mutant by base editor in human embryos

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 （A〉G） mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor （BE） system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 （A〉G） mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 （A〉G） homozygous mutation. Data showed that base editor could precisely correct HBB -28 （A〉G） mutation in the patient＇s primary cells. To model homozygous mutation disease embryos, we consb＇ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured （IVM） oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Phosphorylation of PLIN3 by AMPK promotes dispersion of lipid droplets during starvation

Dear Editor,Lipid droplets(LDs)are dynamic lipid-storage organelles of storage depots and sources of essential substrates for myriad cellular processes and protect cells from lipotoxicity(Ohsaki et al.,2006).Disrupted LD and fat storage homeostasis has been linked to metabolic diseases such as atherosclerosis,obesity,and type II diabetes(Levin et al.,2001).Structurally,the core of neutral lipids in LDs is surroun ded by a phospholipid mono layer and coated with specific proteins(Storey et al.,2011).Perilipin family of proteins are the predominant LD-associated proteins.

Identification of Methylosome Components as Negative Regulators of Plant Immunity Using Chemical Genetics

Nucleotide-binding leucine-rich repeat （NLR） proteins serve as immune receptors in both plants and animals. To identify components required for NLR-mediated immunity, we designed and carried out a chemical genetics screen to search for small molecules that can alter immune responses in Arabidopsis thaliana. From 13 600 compounds, we identified Ro 8-4304 that was able to specifically suppress the severe autoimmune phenotypes of chs3-2D （chilling sensitive 3, 2D）, including the arrested growth morphology and heightened PR （Pathogenesis Related） gene expression. Further, six Ro 8-4304 insensitive mutants were uncovered from the Ro 8-4304-insensitive mutant （rim） screen using a mutagenized chs3-2D popula- tion. Positional cloning revealed thatriml encodes an allele of AtlCIn （I, currents; CI, chloride; n, nucleotide）. Genetic and biochemical analysis demonstrated that AtlCIn is in the same protein complex with the meth- ylosome components small nuclear ribonucleoprotein D3b （SmD3b） and protein arginine methyltransferase 5 （PRMT5）, which are required for the biogenesis of small nuclear ribonucleoproteins （snRNPs） involved in mRNA splicing. Double mutant analysis revealed that SmD3b is also involved in the sensitivity to Ro 8-4304, and the prmt5-1 chs3-2D double mutant is lethal. Loss of At/C/n, SmD3b, or PRMT5 function results in enhanced disease resistance against the virulent oomycete pathogen Hyaloperonospora arabidopsidis Noco2, suggesting that mRNA splicing plays a previously unknown negative role in plant immunity. The successful implementation of a high-throughput chemical genetic screen and the identification of a small-molecule compound affecting plant immunity indicate that chemical genetics is a powerful tool to study whole-organism plant defense pathways.

Bend family proteins mark chromatin boundaries and synergistically promote early germ cell differentiation

Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro.In this study,we developed a coupled screening strategy that took advantage of an arrayed bi-molecular fluorescence complementation(BiFC)platform for protein-protein interaction screens and epiblast-like cell(EpiLC)-induction assays using reporter mouse embryonic stem cells(mESCs).Investigation of candidate interaction partners of core human pluripotent factors OCT4,NANOG,KLF4 and SOX2 in EpiLC differentiation assays identified novel primordial germ cell(PGC)-inducing factors including BEN-domain(BEND/Bend)family members.Through RNA-seq,ChIP-seq,and ATAC-seq analyses,we showed that Bend5 worked together with Bend4 and helped mark chromatin boundaries to promote EpiLC induction in vitro.Our findings suggest that BEND/Bend proteins represent a new family of transcriptional modulators and chromatin boundary factors that participate in gene expression regulation during early germline development.

TPP1 as a versatile player at the ends of chromosomes

Telomeres, the ends of linear eukaryotic chromosomes, are tandem DNA repeats and capped by various telomeric proteins. These nucleoprotein complexes protect telomeres from DNA damage response （DDR）, recombination, and end-to-end fusions, ensuring genome stability. The human telosome/shelterin complex is one of the best-studied telomere-associated protein complexes, made up of six core telomeric proteins TRF1, TRF2, TIN2, RAPI, POT1, and TPPI. TPP1, also known as adrenocortical dysplasia protein homolog （ACD）, is a putative mammalian homolog of TEBP-β and belongs to the oligonucleotide binding （OB）-fold-containing protein family. Three functional domains have been identified within TPP1, the N-terminal OB fold, the POT1 binding recruitment domain （RD）, and the carboxyl-terminal TIN2-interacting domain （TID）. TPP1 can interact with both POT1 and TIN2 to maintain telomere structure, and mediate telomerase recruitment for telomere elongation. These features have indicated TPP1 play an essential role in telomere maintenance. Here, we will review important findings that highlight the functional significance of TPP1, with a focus on its interaction with other telosome components and the telomerase. We will also discuss potential implications in disease therapies.

Cost-effective generation of A-to-G mutant mice by zygote electroporation of adenine base editor ribonucleoproteins

More than 32,000 pathogenic single nucleotide polymorphisms(SNPs)have been identified in the human genome(Gaudelli et al.,2017).Genetically modified mice with pathogenic SNPs are good models for studies of disease pathogenesis and the development of new therapeutics.Accordingly,an efficient,high-throughput method for the generation of mouse models with SNPs is needed.

Effective and precise adenine base editing n mouse zygotes

Dear Editor, Many human genetic diseases are caused by pathogenic single nucleotide mutations. Animal models are often used to study these diseases where the pathogenic point mutations are created and/or corrected through gene editing （e.g., the CRISPP-JCas9 system） （Komor et al., 2017; Liang et al., 2017）. CRISPR/Cas9-mediated gene editing depends on DNA double-strand breaks （DSBs）, which can be of low efficiency and lead to indels and off-target cleavage （Kim et al., 2016）. We and others have shown that base editors （BEs） may represent an attractive alternative for disease mouse model generation （Liang et al., 2017; Kim et al., 2017）. Compared to CRISPR/ Cas9, cytidine base editors （CBEs） can generate C·G to T·A mutations in mouse zygotes without activating DSB repair pathways （Liang et al., 2017; Kim et al., 2017; Komor et al., 2016）. In addition, CBEs showed much lower off-targets than CRISPR]Cas9 （Kim et al., 2017）, making the editing process potentially safer and more controllable. Recently, adenine base editors （ABEs） that were developed from the tRNA- specific adenosine deaminase （TADA） of Escherichia coli were also reported （Gaudelli et al., 2017）. As a RNA-guided programmable adenine deaminase, ABE can catalyze the conversion of A to I. Following DNA replication, base I is replaced by G, resulting in A·T to G·C conversion （Gaudelli et al., 2017; Hu et al., 2018）. The development of ABEs has clearly expanded the editing capacity and application of BEs. Here, we tested whether ABEs could effectively generate disease mouse models, and found high efficiency by ABEs in producing edited mouse zygotes and mice with single-nucleotide substitutions.

Inhibition of tumor suppressor p73 by nerve growth factor receptor via chaperone-mediated autophagy

The tumor suppressr p73 is a homolog of p53 and is capable of inducing cell cycle arrest and apoptosis.Here,we identify nerve growth factor receptor(NGFR,p75NTR,or CD271)as a novel negative p73 regulator.p73 activates NGFR transcription,which,in turn,promotes p73 degradation in a negative feedback loop.NGFR directly binds to p73 central DNA-binding domain and suppresses p73 transcriptional activity as well as p73-mediated apoptosis in cancer cells.Surprisingly,we uncover a previously unknown mechanism of NGFR-facilitated p73 degradation through the chaperone-mediated autophagy(CMA)pathway.Collectively,our studies demonstrate a new oncogenic function for NGFR in inactivating p73 activity by promoting its degradation through the CMA.

Effective gene editing by high-fidelity base editor 2 in mouse zygotes

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical applicaUon of such approaches. Recently, a base editor （BE） system built on cytidine （C） deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine （T） efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high- fidelity version of base editor 2 （HF2-BE2）, and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.

Telomere regulation in pluripotent stem cells

Pluripotent stem cells （PSCs） have the potential to pro- duce any types of cells from all three basic germ layers and the capacity to self-renew and proliferate indefinitely in vitro. The two main types of PSCs, embryonic stem cells （ESCs） and induced pluripotent stem cells （iPSCs）, share common features such as colony morphology, high expression of Oct4 and Nanog, and strong alkaline phosphatase activity. In recent years, increasing evi- dences suggest that telomere length represents another important internal factor in maintaining stem cell pluri- potency. Telomere length homeostasis and its structural integrity help to protect chromosome ends from recom- bination, end fusion, and DNA damage responses, ensuring the divisional ability of mammalian cells. PSCs generally exhibit high telomerase activity to maintain their extremely long and stable telomeres, and emerging data indicate the alternative lengthening of telomeres （ALT） pathway may play an important role in telomere functions too. Such characteristics are likely key to their abilities to differentiate into diverse cell types in v/vo. In this review, we will focus on the function and regulation of telomeres in ESCs and iPSCs, thereby shedding light on the importance of telomere length to pluripotency and the mechanisms that regulate telomeres in PSCs.