Circular RNAs （circRNAs） are recently identified as a naturally occurring family of widespread and diverse endogenous noncoding RNAs that may regulate gene expression in mammals . They are unusually sta- ble RNA molecules with cell type- or developmental stage-specific expression patterns . Exosomes are small membrane vesicles of endocytic origin secreted by most cell types. They contain a specific cargo of protein, mRNA and microRNA species, which can modulate recipient cell behaviors and may be used as biomarkers for diagnosis of human diseases .
Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N-6-methyladenosine (m(6)A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m(6)A motifs are enriched in circRNAs and a single m(6)A site is sufficient to drive translation initiation. This m(6)A-driven translation requires initiation factor eIF4G2 and m(6)A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m(6)A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.
Exosomes, small membrane vesicles （30-100 nm） of endocytic origin secreted by most cell types, contain functional biomolecules, which can be horizontally transferred to recipient cells . Exosomes bear a specific protein and lipid composition, and carry a select set of functional mRNAs and microRNAs . Recently, our group has shown that c-Met shed in exosomes can promote a proangiogenic and prometastatic phenotype in bone marrow-derived progenitor cells during melanoma progression . In previous research, retrotransposon RNA transcripts, single-stranded DNA （ssDNA）,
Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1 beta (IL-1 beta) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1 beta in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1 beta secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd(-/-) cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd(-/-) cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.
Dear Editor, In the past few years, the development of sequence- specific DNA nucleases has progressed rapidly and such nucleases have shown their power in generating efficient targeted mutagenesis and other genome editing applica- tions. For zinc finger nucleases （ZFNs） and transcription activator-like effector nucleases （TALENs）, an engi- neered array of sequence-specific DNA binding domains are fused with the DNA nuclease Fokl [1, 2]. These nu- cleases have been successful in genome modifications by generating double strand breaks （DSBs）, which are then repaired through non-homologous end joining （NHEJ） or homologous recombination （HR） in different species, including mouse, tobacco and rice [3-5]. Recently, an- other breakthrough technology for genome editing, the CRISPR/Cas system, was developed. CRISPR （clustered regulatory interspaced short 12alindromic repeats） loci are variable short spacers separated by short repeats, which are transcribed into non-coding RNAs. The non-coding RNAs form a functional complex with CRISPR-asso- ciated （Cas） proteins and guide the complex to cleave complementary invading DNA . After the initial development of a programmable CRISPR/Cas system, it has been rapidly applied to achieve efficient genome editing in human cell lines, zebrafish and mouse [7-10]. However, there is still no successful application in plants reported.
The methyltransferase like 3 （METTL3）-containing methyltransferase complex catalyzes the N6-methyladenosine （m6A） formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms＇ tumor 1-associating protein （WTAP） and methyltransferase like 14 （METTL14）. WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic ac- tivity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-en- hanced crosslinking and immunoprecipitation （PAR-CLIP） illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.
Recent advances with the type II clustered regularly interspaced short palindromic repeats （CRISPR） system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site- specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrpv11 mutant embryos or cardia bifida phenotypes in fautm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, envi- ronmental studies and medicine.
Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 （dCas9） protein fused with a transcriptional activation domain and single guide RNAs （sgRNAs） with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.
Genome editing of model organisms is essential for gene function analysis and is thus critical for human health and agricultural production. The current technolo- gies used for genome editing include ZFN （zinc-finger nuclease）, meganucleases, TALEN （Transcription activa- tor-like effector nucleases）, etc. . These technologies can generate double stranded breaks （DSBs） to either disrupt gene function through generation of premature stop codons by non-homologous end joining （NHEJ） pathway, or to facilitate gene targeting through homolo- gous recombination （HR） with an incoming template. Recently, a new technology for genome editing, CRISPR （Clustered Regularly Interspaced Short Palindromic Re- peats）/Cas （CRISPR-associated） systems, has been de- veloped . CRISPR/Cas systems are adaptive defense systems in prokaryotic organisms to fight against alien nucleic acids . The spacer sequences acquired from foreign DNA are positioned between host repeats, and transcribed together as CRISPR RNA （crRNA）. In the type II CRISPR system, a single nuclease Cas9, guided by a dual-crRNA：tracrRNA, is sufficient to cleave cog- nate DNA homologous to the spacer . Efficient cleav- age also requires the presence of protospacer adjacent motif （PAM） 5＇-NGG-3＇ following the spacer sequence. The dual-crRNA：tracrRNA has been further streamlined to a single RNA chimera, called sgRNA （single guide RNA） . Compared with protein-guided technologies, CRISPR/Cas system is much easier to implement, as only short guide RNAs need to be customized to target the genes of interest. Up to now, the CRISPR/Cas sys- tem has been successfully applied to efficient genome editing in many eukaryotic organisms including human , mice , zebra fish , fly , worm , and yeast . However, the application of CRISPR/Cas system in plants has not been reported. Rice （Oryza sativa L.） is a major staple crop in the grass family （Poaceae）, feed- ing half of the world＇s population.
The human 8q24 gene desert contains multiple enhancers that form tissue-specific long-range chromatin loops with the MYC oncogene, but how chromatin looping at the MYC locus is regulated remains poorly understood. Here we demonstrate that a long noncoding RNA (lncRNA), CCAT1-L, is transcribed specifically in human colorectal cancers from a locus 515 kb upstream of MYC. This lncRNA plays a role in MYC transcriptional regulation and promotes long-range chromatin looping. Importantly, the CCAT1-L locus is located within a strong super-enhancer and is spatially close to MYC. Knockdown of CCAT1-L reduced long-range interactions between the MYC promoter and its enhancers. In addition, CCAT1-L interacts with CTCF and modulates chromatin conformation at these loop regions. These results reveal an important role of a previously unannotated lncRNA in gene regulation at the MYC locus.
The role of Fat Mass and Obesity-associated protein （FTO） and its substrate N6-methyladenosine （m6A） in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adi- pogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5＇- and 3＇-splice sites, spatially over- lapping with mRNA splicing regulatory serine/arginine-rich （SR） protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenie regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.
N-6-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in cell differentiation and tissue development. It regulates multiple steps throughout the RNA life cycle including RNA processing, translation, and decay, via the recognition by selective binding proteins. In the cytoplasm, m(6)A binding protein YTHDF1 facilitates translation of m(6)A-modified mRNAs, and YTHDF2 accelerates the decay of m(6)A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m(6)A binder of the YTH (YT521-B homology) domain family, remains unknown. Here, we report that YTHDF3 promotes protein synthesis in synergy with YTHDF1, and affects methylated mRNA decay mediated through YTHDF2. Cells deficient in all three YTHDF proteins experience the most dramatic accumulation of m(6)A-modified transcripts. These results indicate that together with YTHDF1 and YTHDF2, YTHDF3 plays critical roles to accelerate metabolism of m(6)A-modified mRNAs in the cytoplasm. All three YTHDF proteins may act in an integrated and cooperative manner to impact fundamental biological processes related to m(6)A RNA methylation.
Mixed lineage kinase domain-like protein （MLKL） was identified to function downstream of receptor interacting protein 3 （RIP3） in tumor necrosis factor-α （TNF）-induced necrosis （also called necroptosis）. However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain （HBD＊） triggers necroptosis. Notably, forcing together the N-terminal domain （ND） but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL （1-130 amino acids） is sufficient to trigger necroptosis. Both the HBD＊-mediated and TNF-induced complexes of MLKL（ND） or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plas- ma membrane location is located in the junction of the first and second a-helices of MLKL. The plasma membrane translocation of MLKL or MLKL（ND） leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture.
Our previous studies have demonstrated that stable microRNAs (miRNAs) in mammalian serum and plasma are actively secreted from tissues and cells and can serve as a novel class of biomarkers for diseases, and act as signaling molecules in intercellular communication. Here, we report the surprising finding that exogenous plant miRNAs are present in the sera and tissues of various animals and that these exogenous plant miRNAs are primarily acquired orally, through food intake. MIR168a is abundant in rice and is one of the most highly enriched exogenous plant miRNAs in the sera of Chinese subjects. Functional studies in vitro and in vivo demonstrated that MIR168a could bind to the human/ mouse low-density lipoprotein receptor adapter protein 1 (LDLRAP1) mRNA, inhibit LDLRAP1 expression in liver, and consequently decrease LDL removal from mouse plasma. These findings demonstrate that exogenous plant miRNAs in food can regulate the expression of target genes in mammals.
The recent Zika virus （ZIKV） epidemic in Latin America coincided with a marked increase in microcephaly in newborns. However, the causal link between maternal ZIKV infection and malformation of the fetal brain has not been firmly established. Here we show a vertical transmission of ZIKV in mice and a marked effect on fetal brain development. We found that intraperitoneal （i.p.） injection of a contemporary ZIKV strain in pregnant mice led to the infection of radial gila cells （RGs） of dorsal ventricular zone of the fetuses, the primary neural progenitors responsi- ble for cortex development, and caused a marked reduction of these cortex founder cells in the fetuses. Interestingly, the infected fetal mice exhibited a reduced cavity of lateral ventricles and a discernable decrease in surface areas of the cortex. This study thus supports l;he conclusion that vertically transmitted ZIKV affects fetal brain development and provides a valuable animal model for the evaluation of potential therapeutic or preventative strategies.
Dysregulated expression of microRNAs （miRNAs） in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.
The pig is an important livestock for food supply and an ideal model for various human diseases. Efficient and precise genetic engineering in pigs holds great promise in agriculture and biomedicine . Using currently available approach, generating specific gene modifications in pigs requires two steps. First, site-specific nucleases such as zinc finger nucleases （ZFNs） and transcription activator-like effector nucleases （TALENs） are used to generate targeted mutations in pig somatic cells.
Although the function of DNA methylation in gene promoter regions is well established in transcriptional repression, the function of the evolutionarily conserved widespread distribution of DNA methylation in gene body regions remains incompletely understood. Here, we show that DNA methylation is enriched in included alternatively spliced exons （ASEs）, and that inhibition of DNA methylation results in aberrant splicing of ASEs. The methyl- CpG-binding protein MeCP2 is enriched in included ASEs, particularly those that are also highly methylated, and inhibition of DNA methylation disrupts specific targeting of MeCP2 to exons. Interestingly, ablation of MeCP2 results in increased histone acetylation and aberrant ASE-skipping events. We further show that inhibition of histone deacetylase （HDAC） activity leads to exon skipping that shows a highly significant degree of overlap with that caused by MeCP2 knockdown. Together, our data indicate that intragenic DNA methylation operates in exon definition to modulate alternative RNA splicing and can enhance exon recognition via recruitment of the multifunctional protein MeCP2, which thereby maintains local histone hypoacetylation through the subsequent recruitment of HDACs.
Single-ceU genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple om- ics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations （CNVs）, DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs gen- erally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.