Showing papers in "Cell Research in 2018"

Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism.

Abstract: N6-methyladenosine (m6A) is a chemical modification present in multiple RNA species, being most abundant in mRNAs. Studies on enzymes or factors that catalyze, recognize, and remove m6A have revealed its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. This review describes the current understanding of the m6A modification, particularly the functions of its writers, erasers, readers in RNA metabolism, with an emphasis on its role in regulating the isoform dosage of mRNAs.

Mitochondrial metabolism and cancer.

Abstract: Glycolysis has long been considered as the major metabolic process for energy production and anabolic growth in cancer cells. Although such a view has been instrumental for the development of powerful imaging tools that are still used in the clinics, it is now clear that mitochondria play a key role in oncogenesis. Besides exerting central bioenergetic functions, mitochondria provide indeed building blocks for tumor anabolism, control redox and calcium homeostasis, participate in transcriptional regulation, and govern cell death. Thus, mitochondria constitute promising targets for the development of novel anticancer agents. However, tumors arise, progress, and respond to therapy in the context of an intimate crosstalk with the host immune system, and many immunological functions rely on intact mitochondrial metabolism. Here, we review the cancer cell-intrinsic and cell-extrinsic mechanisms through which mitochondria influence all steps of oncogenesis, with a focus on the therapeutic potential of targeting mitochondrial metabolism for cancer therapy.

Plasma membrane changes during programmed cell deaths

Abstract: Ruptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death, respectively. As such, apoptosis is usually considered a non-inflammatory process while necrosis triggers inflammation. Recent studies on necroptosis and pyroptosis, two types of programmed necrosis, revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D (GSDMD) pores during pyroptosis. Importantly, the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores. Interestingly, it was found recently that secondary necrosis of apoptotic cells, a previously believed non-regulated form of cell lysis that occurs after apoptosis, can be programmed and executed by plasma membrane pore formation like that of pyroptosis. In addition, pyroptosis is associated with pyroptotic bodies, which have some similarities to apoptotic bodies. Therefore, different cell death programs induce distinctive reshuffling processes of the plasma membrane. Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity, not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response. In this review, we will discuss recent advances in the field of apoptosis, necroptosis and pyroptosis, with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their implication in cell death-elicited immunogenicity.

CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA.

Abstract: Dear Editor, The CRISPR-associated protein Cas12a (previously known as Cpf1), which is an endonuclease from the type V-A CRISPR system, has been applied in both in vivo genome editing and in vitro DNA assembly. Cas12a is guided by a single CRISPR RNA (crRNA) with a T-rich protospacer adjacent motif (PAM) sequence to cleave double-stranded DNA (dsDNA) targets, generating sticky ends. Different from Cas9, Cas12a cleaves both the target and non-target strands of a targeted dsDNA by a single active site in the RuvC catalytic pocket (Supplementary information, Figure S12a). Besides, Cas12a also processes precursor crRNAs to generate mature crRNAs. However, the cleavage activity of Cas12a on single-stranded DNA (ssDNA) targets is less understood. To investigate the ssDNA cleavage feature of Cas12a, we employed FnCas12a to cleave short ssDNAs that were labelled with 5(6)-carboxyfluorescein (FAM) on the 3′ terminus and found that the ssDNA cleavage sites were near the 22nd base (i.e., from the 21st to the 23rd), counting from the first 3′-base that was paired with the crRNA guide sequence (Supplementary information, Figure S1a and b and Tables S2 and 3). The cleavage did not require the existence of a PAM sequence in the targeted ssDNA (Supplementary information, Figure S1a and b). In addition, the same cleavage sites were obtained with crRNAs having guide sequences as short as 10 nucleotides (nt) (Supplementary information, Figure S1c and d), which indicates that Cas12a could cleave ssDNA at sites outside of the recognition sequence. We then tested Cas12a cleavage efficiency on ssDNA and dsDNA substrates, and cleavage of ssDNA was slower than that of dsDNA (Supplementary information, Figure S1e and f), whose PAM sequence may account for the higher efficiency. We also performed the Cas12a cleavage experiment with a ssDNA target labelled at its 5′ terminus (target-DNMT1-3-R-FAM5′). Surprisingly, no cleaved bands were observed at the predicted size (20 nt), but short (<6 nt) FAM-labelled products were generated (Fig. 1a). After careful analyses of experimental conditions, we found that only the ternary complex of Cas12a/ crRNA/targeted ssDNA (or targeted dsDNA) was able to cleave the 5′-labelled target ssDNA (target-DNMT1-3), generating short FAM-labelled products (Fig. 1b and Supplementary information, Figure S2). The ternary complex also promiscuously cleaved collateral ssDNAs that had no complementarity to the crRNA guide sequence in the reaction system, generating short products (Fig. 1c and Supplementary information, Figure S3). As it is difficult to distinguish the precise length of the short transcleavage products via polyacrylamide gel electrophoresis, the FAM-labelled short products were purified and analysed by liquid chromatography-mass spectrometry. The results showed that 5′-FAM-labelled substrates were mainly trans-cleaved to 4 nt, while 2-nt products were observed for 3′-FAM-labelled substrates (Supplementary information, Figure S4). We called the promiscuous cleavage of collateral ssDNAs transcleavage to distinguish it from the programmable on-target cleavage of target ssDNA (namely, cis-cleavage), and the proposed ssDNA cleavage processes were illustrated in Fig. 1d. When the ssDNA substrate was labelled at the 5′ terminus, the cis-cleaved 5′-labelled ssDNA products became collateral ssDNAs in the reaction system and were subsequently trans-cleaved into short products, explaining the observed cleavage pattern for 5′-labelled ssDNA substrate. We observed the trans-cleavage products in addition to the cis-products for short 3′-labelled targeted ssDNAs (Fig. 1a). The majority of the ternary complex most likely remained bound to the targeted ssDNAs after cis-cleavage, protecting the labelled 3′-terminus from exposing the trans-activity sites of the Cas12a ternary complex. Next, we tested nine randomly selected Cas12a proteins from different species in addition to the above tested FnCas12a (Supplementary information, Figures S5, 6a and Tables S1, 4 and 5), and all Cas12a proteins exhibited endonuclease activity on plasmid dsDNA (Supplementary information, Figure S6b), cis(Supplementary information, Figure S6c) and trans-cleavage activities on ssDNA (Supplementary information, Figure S6d). This indicates that the cisand trans-cleavage activities on ssDNA might be ubiquitous among Cas12a proteins. When shortened targeted ssDNAs were tested, complexes with 18-nt target ssDNAs that lacked a cleavage site also showed transcleavage activity (Supplementary information, Figure S7a), indicating that cis-cleavage was not a prerequisite for trans-cleavage activity. Trans-cleavage was implemented by the endonuclease activity of the complex, as circular ssDNA (M13mp18) could also be trans-cleaved (Supplementary information, Figure S7b). Moreover, we found that all tested Cas12a complexes except the AsCas12a complex had trans-cleavage activity on collateral dsDNAs (Figure S8), and the activity of the LbCas12a, BoCas12a and Lb4Cas12a complexes was much higher. To identify key residues involved in ssDNA cleavage of both targeted and collateral ssDNAs, we mutated several candidate residues in FnCas12a to alanines, including those related to the RNase activity (H843, K852 and K869) and those responsible for dsDNA cleavage (D917, E1006, D1255 and R1218) (refs. 1,7–9 and Supplementary information, Figure S9). Both cisand transcleavage of ssDNA were unaffected in the RNase activity-related mutants (Supplementary information, Figure S10), but the activities were completely lost or remarkably decreased with mutations in either the RuvC domain (D917A, E1006A and D1255A mutations in FnCas12a) or the Nuc domain (R1218A mutation in FnCas12a) (Fig. 1e, f). Recent studies showed that the RuvC catalytic pocket of both C2c1 and Cas12a was responsible for the cleavage of both strands of targeted dsDNA, leading us to propose that targeted ssDNAs were cis-cleaved by this catalytic pocket (Supplementary information, Figures S11a, b, d, e and S12c). Moreover, according to the structure of the C2c1-crRNAexcess DNA complex (Supplementary information, Figure S11c), trans-cleavage of collateral ssDNAs could also be achieved by the same catalytic pocket (Fig. 1g, and Supplementary information,

RNA N6-methyladenosine modification in cancers: current status and perspectives

Abstract: N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), has been shown to play critical roles in various normal bioprocesses such as tissue development, stem cell self-renewal and differentiation, heat shock or DNA damage response, and maternal-to-zygotic transition. The m6A modification is deposited by the m6A methyltransferase complex (MTC; i.e., writer) composed of METTL3, METTL14 and WTAP, and probably also VIRMA and RBM15, and can be removed by m6A demethylases (i.e., erasers) such as FTO and ALKBH5. The fates of m6A-modified mRNAs rely on the functions of distinct proteins that recognize them (i.e., readers), which may affect the stability, splicing, and/or translation of target mRNAs. Given the functional importance of the m6A modification machinery in normal bioprocesses, it is not surprising that evidence is emerging that dysregulation of m6A modification and the associated proteins also contributes to the initiation, progression, and drug response of cancers. In this review, we focus on recent advances in the study of biological functions and the underlying molecular mechanisms of dysregulated m6A modification and the associated machinery in the pathogenesis and drug response of various types of cancers. In addition, we also discuss possible therapeutic interventions against the dysregulated m6A machinery to treat cancers.

The adult human testis transcriptional cell atlas

Abstract: Human adult spermatogenesis balances spermatogonial stem cell (SSC) self-renewal and differentiation, alongside complex germ cell-niche interactions, to ensure long-term fertility and faithful genome propagation. Here, we performed single-cell RNA sequencing of ~6500 testicular cells from young adults. We found five niche/somatic cell types (Leydig, myoid, Sertoli, endothelial, macrophage), and observed germline-niche interactions and key human-mouse differences. Spermatogenesis, including meiosis, was reconstructed computationally, revealing sequential coding, non-coding, and repeat-element transcriptional signatures. Interestingly, we identified five discrete transcriptional/developmental spermatogonial states, including a novel early SSC state, termed State 0. Epigenetic features and nascent transcription analyses suggested developmental plasticity within spermatogonial States. To understand the origin of State 0, we profiled testicular cells from infants, and identified distinct similarities between adult State 0 and infant SSCs. Overall, our datasets describe key transcriptional and epigenetic signatures of the normal adult human testis, and provide new insights into germ cell developmental transitions and plasticity.

UPRmt regulation and output: a stress response mediated by mitochondrial-nuclear communication

Abstract: The mitochondrial network is not only required for the production of energy, essential cofactors and amino acids, but also serves as a signaling hub for innate immune and apoptotic pathways. Multiple mechanisms have evolved to identify and combat mitochondrial dysfunction to maintain the health of the organism. One such pathway is the mitochondrial unfolded protein response (UPRmt), which is regulated by the mitochondrial import efficiency of the transcription factor ATFS-1 in C. elegans and potentially orthologous transcription factors in mammals (ATF4, ATF5, CHOP). Upon mitochondrial dysfunction, import of ATFS-1 into mitochondria is reduced, allowing it to be trafficked to the nucleus where it promotes the expression of genes that promote survival and recovery of the mitochondrial network. Here, we discuss recent findings underlying UPRmt signal transduction and how this adaptive transcriptional response may interact with other mitochondrial stress response pathways.

Amyloid fibril structure of α-synuclein determined by cryo-electron microscopy.

Abstract: α-Synuclein (α-syn) amyloid fibrils are the major component of Lewy bodies, which are the pathological hallmark of Parkinson’s disease (PD) and other synucleinopathies. High-resolution structure of α-syn fibril is important for understanding its assembly and pathological mechanism. Here, we determined a fibril structure of full-length α-syn (1–140) at the resolution of 3.07 A by cryo-electron microscopy (cryo-EM). The fibrils are cytotoxic, and transmissible to induce endogenous α-syn aggregation in primary neurons. Based on the reconstructed cryo-EM density map, we were able to unambiguously build the fibril structure comprising residues 37–99. The α-syn amyloid fibril structure shows two protofilaments intertwining along an approximate 21 screw axis into a left-handed helix. Each protofilament features a Greek key-like topology. Remarkably, five out of the six early-onset PD familial mutations are located at the dimer interface of the fibril (H50Q, G51D, and A53T/E) or involved in the stabilization of the protofilament (E46K). Furthermore, these PD mutations lead to the formation of fibrils with polymorphic structures distinct from that of the wild-type. Our study provides molecular insight into the fibrillar assembly of α-syn at the atomic level and sheds light on the molecular pathogenesis caused by familial PD mutations of α-syn.

Exosomal PD-L1 harbors active defense function to suppress T cell killing of breast cancer cells and promote tumor growth.

Abstract: Dear Editor, The tumor-microenvironment interactions play important roles in tumor progression, metastasis, and therapeutic resistance, and increasing evidence indicates that tumor cellderived exosomes can systematically modulate or reprogram the tumor microenvironment by transferring molecules, such as microRNAs, mRNAs, and proteins from donor cells to recipient cells. PD-L1 is a classical immune surface protein, which inhibits anti-tumor function of T cells by binding to its receptor programmed cell death-1 (PD-1) and effectively protects tumor from immune surveillance. Exosomes have been reported to contain certain types of proteins, including membrane proteins, e.g., EGFR and MET, that promote cancer metastasis. As a membrane-bound protein, whether PD-L1 exists in cancer cellderived exosomes and whether it plays a role in tumor progress are largely unknown. We isolated exosomes from the supernatant of cell cultures of MDA-MB-231 (231) human breast cancer cells and 4T1 mouse mammary tumor cells with PD-L1 expression or PD-L1 knockout (PD-L1) by sequential centrifugation. Transmission electron microscopy images showed that these exosomes are typically spherical and membrane encapsulated with a size of 30–100 nm (Supplementary information, Figure S1a). PD-L1 was detected in exosomes isolated from the culture media of PD-L1-expressing human breast cancer cells (231-PD-L1) and mouse mammary tumor cells (4T1-PD-L1), but not 231-PD-L1 or 4T1-PD-L1 cells with similar levels of exosome makers, CD63 and CD81, whose expression indicates exosome production (Fig. 1a; Supplementary information, Figure S1b and c). Notably, treatment with exosome secretion inhibitor, GW4869, reduced exosome production (as indicated by the reduction of exosome markers CD63 and CD81 or the total amount of exosomal protein) in 231 cells as well as the levels of PD-L1 in exosomes, but had no effect on PD-L1 expression in the cell lysates (Fig. 1b; Supplementary information, Figure S1d). In addition, in vitro binding assay showed that PD-1Fc protein simultaneously pulled down PD-L1 and CD81 in 231PD-L1-derived exosomes (term as exosome-PD-L1) (Fig. 1c). Immunofluorescence (IF) staining of 231 cells (Fig. 1d) and immunohistochemistry (IHC) double staining of human breast cancer tissues (Supplementary information, Figure S1e) demonstrated co-localization of PD-L1 and CD63 in the multivesicular bodies (MVBs), which are the precursor form of exosomes inside cells before released. These data further supported the presence of PD-L1 in exosomes in vitro and in vivo. To evaluate the biological functions of exosome-PD-L1, we first asked whether it could transfer PD-L1 to other cells with low (MCF7) or no PD-L1 expression (BT549-PD-L1). We detected the transfer of PD-L1 from exosome-PD-L1 to MCF7 or BT549-PD-L1 cells but not from exosomes derived from 231-PD-L1 cells (exosome-PD-L1; Fig. 1e). The acquisition of PD-L1 protein was not a result of PD-L1 gene expression as indicated by the lack of PD-L1mRNA in these cells (Supplementary information, Figure S1f). We also established 231-PD-L1 cells and visually demonstrated the transfer of PD-L1 from 231-PD-L1-derived exosomes to BT549 cells (Supplementary information, Figure S1g). To examine whether this also occurs in vivo, we co-injected mouse 4T1-PDL1 cells with exosomes derived from 4T1-PD-L1 (EX-PDL1), 4T1-PD-L1 (EX-PD-L1), or PBS into the mammary fat pad of BALB/c mice. Tumors were harvested after 5 days. IF staining of tumor tissue sections showed that EX-PD-L1 but not EX-PD-L1 rendered 4T1-PD-L1 cells PD-L1 positive (Supplementary information, Figure S1h). Importantly, results from flow cytometric analysis further revealed that the PD-L1 transported by exosomes was located on the surface of target cells and able to bind to PD-1 (Supplementary information, Figure S1i). These results indicated that exosomes are capable of transferring functional PD-L1 to other cells. Given that PD-L1 of exosomes can directly bind to PD-1 (Fig. 1c), we next examined whether exosomal PD-L1 affects T cell functions. As shown in Fig. 1f, exosome-PD-L1, but not exosome-PD-L1 or PBS, significantly inhibited the T cell killing effect on BT549-PD-L1 cells. Next, to explore whether exosomal PD-L1 inhibits CD3/CD28-triggered T cell activation signaling pathway, we generated T cell blasts by treating peripheral blood mononuclear cells (PBMCs) with phytohemagglutinin (PHA) to induce PD-1 expression. The results showed that exosome-PD-L1 but not exosome-PD-L1 markedly inhibited CD3/CD28-induced ERK phosphorylation and NF-κB activation of T cells in a dosedependent manner (Supplementary information, Figure S2a and b) as well as PHA-induced interleukin-2 (IL-2) secretion (Supplementary information, Figure S2c), all of which are indicators of T cell activation. Furthermore, exosomal PD-L1 from other cancer cell lines such as colon (RKO) and lung (HCC827) cancer cells has similar function in blocking T cell activation (IL-2 production; Supplementary information, Figure S2d and e). Together, these data supported that exosomal PD-L1 inhibits T-cell activation. Next, to evaluate the role of exosomal PD-L1 in tumor microenvironment and tumor progression in vivo, we measured tumor growth of 4T1-PD-L1 cells co-injected with EX-PD-L1, EX-PD-L1, or PBS. Consistent with the previous report, PD-L1 deficiency in 4T1-PD-L1 cells led to substantial tumor regression; however, EX-PD-L1 but not EX-PD-L1 remarkably restored tumor growth of 4T1-PD-L1 cells (Fig. 1g). We then exposed 4T1PD-L1 cells to increasing amounts of EX-PD-L1 and showed that EX-PD-L1 promoted tumor growth in a dose-dependent manner (Supplementary information, Figure S2f). Moreover, 4T1PD-L1 tumors with EX-PD-L1 co-injection exhibited much less granzyme B expression, indicating reduced cytotoxic T cell activity, in tumor area compared with those with EX-PD-L1 or PBS co-

Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

Abstract: Iron has been shown to trigger oxidative stress by elevating reactive oxygen species (ROS) and to participate in different modes of cell death, such as ferroptosis, apoptosis and necroptosis. However, whether iron-elevated ROS is also linked to pyroptosis has not been reported. Here, we demonstrate that iron-activated ROS can induce pyroptosis via a Tom20-Bax-caspase-GSDME pathway. In melanoma cells, iron enhanced ROS signaling initiated by CCCP, causing the oxidation and oligomerization of the mitochondrial outer membrane protein Tom20. Bax is recruited to mitochondria by oxidized Tom20, which facilitates cytochrome c release to cytosol to activate caspase-3, eventually triggering pyroptotic death by inducing GSDME cleavage. Therefore, ROS acts as a causative factor and Tom20 senses ROS signaling for iron-driven pyroptotic death of melanoma cells. Since iron activates ROS for GSDME-dependent pyroptosis induction and melanoma cells specifically express a high level of GSDME, iron may be a potential candidate for melanoma therapy. Based on the functional mechanism of iron shown above, we further demonstrate that iron supplementation at a dosage used in iron-deficient patients is sufficient to maximize the anti-tumor effect of clinical ROS-inducing drugs to inhibit xenograft tumor growth and metastasis of melanoma cells through GSDME-dependent pyroptosis. Moreover, no obvious side effects are observed in the normal tissues and organs of mice during the combined treatment of clinical drugs and iron. This study not only identifies iron as a sensitizer amplifying ROS signaling to drive pyroptosis, but also implicates a novel iron-based intervention strategy for melanoma therapy.

Polyubiquitin chain-induced p62 phase separation drives autophagic cargo segregation

Abstract: Misfolded proteins can be degraded by selective autophagy. The prevailing view is that ubiquitin-tagged misfolded proteins are assembled into aggregates by the scaffold protein p62, and the aggregates are then engulfed and degraded by autophagosomes. Here we report that p62 forms droplets in vivo which have liquid-like properties such as high sphericity, the ability to undergo fusion, and recovery after photobleaching. Recombinant p62 does not undergo phase separation in vitro; however, adding a K63 polyubiquitin chain to p62 induces p62 phase separation, which results in enrichment of high-molecular weight ubiquitin signals in p62 droplets. Mixing recombinant p62 with cytosol from p62−/− cells also results in p62 phase separation in a polyubiquitination-dependent manner. Mechanistically, p62 phase separation is dependent on p62 polymerization, the interaction between p62 and ubiquitin, and the valence of the polyubiquitin chain. Moreover, p62 phase separation can be regulated by post-translational modifications such as phosphorylation. Finally, we demonstrate that disease-associated mutations in p62 can affect phase separation. We propose that polyubiquitin chain-induced p62 phase separation drives autophagic cargo concentration and segregation.

Modifications and functional genomics of human transfer RNA.

Abstract: Transfer RNA (tRNA) is present at tens of millions of transcripts in a human cell and is the most abundant RNA in moles among all cellular RNAs. tRNA is also the most extensively modified RNA with, on an average, 13 modifications per molecule. The primary function of tRNA as the adaptor of amino acids and the genetic code in protein synthesis is well known. tRNA modifications play multi-faceted roles in decoding and other cellular processes. The abundance, modification, and aminoacylation (charging) levels of tRNAs contribute to mRNA decoding in ways that reflect the cell type and its environment; however, how these factors work together to maximize translation efficiency remains to be understood. tRNAs also interact with many proteins not involved in translation and this may coordinate translation activity and other processes in the cell. This review focuses on the modifications and the functional genomics of human tRNA and discusses future perspectives on the explorations of human tRNA biology.

Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis.

Abstract: A systematic interrogation of male germ cells is key to complete understanding of molecular mechanisms governing spermatogenesis and the development of new strategies for infertility therapies and male contraception. Here we develop an approach to purify all types of homogeneous spermatogenic cells by combining transgenic labeling and synchronization of the cycle of the seminiferous epithelium, and subsequent single-cell RNA-sequencing. We reveal extensive and previously uncharacterized dynamic processes and molecular signatures in gene expression, as well as specific patterns of alternative splicing, and novel regulators for specific stages of male germ cell development. Our transcriptomics analyses led us to discover discriminative markers for isolating round spermatids at specific stages, and different embryo developmental potentials between early and late stage spermatids, providing evidence that maturation of round spermatids impacts on embryo development. This work provides valuable insights into mammalian spermatogenesis, and a comprehensive resource for future studies towards the complete elucidation of gametogenesis.

Tissue clearing of both hard and soft tissue organs with the PEGASOS method.

Abstract: Tissue clearing technique enables visualization of opaque organs and tissues in 3-dimensions (3-D) by turning tissue transparent. Current tissue clearing methods are restricted by limited types of tissues that can be cleared with each individual protocol, which inevitably led to the presence of blind-spots within whole body or body parts imaging. Hard tissues including bones and teeth are still the most difficult organs to be cleared. In addition, loss of endogenous fluorescence remains a major concern for solvent-based clearing methods. Here, we developed a polyethylene glycol (PEG)-associated solvent system (PEGASOS), which rendered nearly all types of tissues transparent and preserved endogenous fluorescence. Bones and teeth could be turned nearly invisible after clearing. The PEGASOS method turned the whole adult mouse body transparent and we were able to image an adult mouse head composed of bones, teeth, brain, muscles, and other tissues with no blind areas. Hard tissue transparency enabled us to reconstruct intact mandible, teeth, femur, or knee joint in 3-D. In addition, we managed to image intact mouse brain at sub-cellular resolution and to trace individual neurons and axons over a long distance. We also visualized dorsal root ganglions directly through vertebrae. Finally, we revealed the distribution pattern of neural network in 3-D within the marrow space of long bone. These results suggest that the PEGASOS method is a useful tool for general biomedical research.

PROTAC-induced BTK degradation as a novel therapy for mutated BTK C481S induced ibrutinib-resistant B-cell malignancies

Abstract: Dear Editor, Non-Hodgkin’s lymphoma (NHL) is a type of cancer that mainly develops from B-cell malignancies, causing 231,400 deaths in 2015 globally. According to the American Cancer Society, around 66,000 new cases of NHL are diagnosed each year in the United States. Bruton’s tyrosine kinase (BTK) is an enzyme encoded by the BTK gene in human. BTK is expressed in all cell lineages of the hematopoietic system except for T cells. As a cytoplasmic tyrosine kinase of the TEC family, BTK plays a crucial role in B cell development, differentiation, and signaling. BTK is closely associated with chronic B-cell receptor (BCR) activation, and is critical for the survival of B-cell neoplasms. Inhibition of BTK kinase activity has been proven to be an important and practical way of treating NHL. Ibrutinib is a first-inclass, covalent BTK inhibitor that is able to bind C481 (Cysteine481) of BTK with an ideal IC50 of 0.5 nM. 4,5 Ibrutinib was approved by the FDA in 2013 to 2015 for the treatment of several types of NHL, specifically relapsed/refractory mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), and Waldenström macroglobulinaemia (WM). Currently, there is an ongoing clinical trial on ibrutinib for its efficacy in DLBCL treatment. However, resistance to ibrutinib has been reported in various lymphomas, including CLL and MCL, due to a C481S (cysteine to serine mutation at position 481) BTK mutation. Because ibrutinib cannot form a covalent bond with the hydroxyl group of serine, C481S mutation increases the IC50 against BTK-C481S phosphorylation from 2.2 nM to 1 μM. Thus, there is an urgent need to develop a new strategy against C481S mutation-induced resistance. In addition, ibrutinib has been reported to induce a variety of side effects, including arthralgias, myalgias, atrial fibrillation, ecchymosis, and major hemorrhage. As ibrutinib shows inhibition of EGFR, ITK and TEC family kinases, with IC50s of around 10–100 nM, the pathogeny could be correlated to these known off-target effects of ibrutinib. Proteolysis-targeting chimera (PROTAC) has emerged as a novel chemical approach for the selective degradation of cellular proteins, known as chemical knockdown of a protein of interest. PROTAC molecules (PROTACs) are small molecules capable of bringing a target protein into the proximity of an E3 ligase of interest, causing consequent degradation of the target protein (Fig. 1a). These heterobifunctional molecules consist of three components: a target protein-binding moiety (targeting arm, TA), a degradation machinery-recruiting unit (degradation arm, DA), and a linker that couples these two functionalities. Typically, the utilized degradation machinery is the ubiquitin–proteasome system (UPS) that recruits an E3 ubiquitin ligase followed by ubiquitination of the target protein and its subsequent degradation by the proteasome. In 2015, Crews and Bradner independently reported that BRD4, which has been implicated in several different cancers, could be efficiently degraded through the PROTAC technique. This presented the PROTAC technique as a promising alternative approach against cancer. Unlike traditional drugs, PROTACs aim to eliminate proteins with aberrant functions, rather than inhibiting their activity. Therefore, the acquired resistance caused by the C481S BTK mutant could, in principle, be overcome by using PROTAC molecules. In this study, for the first time, we report the development of BTK-targeting degraders using the PROTAC strategy. These PROTACs could efficiently degrade ibrutinib-sensitive BTK-WT (wild type). More importantly, our newly designed PROTACs also significantly induced the degradation of ibrutinib-resistant BTK-C481S (50% degradation efficiency at 30 nM). Furthermore, our PROTAC molecules efficiently inhibited cell proliferation and colony formation, while exhibited no obvious inhibition (>1000 nM) of ITK, EGFR, and TEC, which are major off-targets of ibrutinib. These data demonstrate the strong potential for developing PROTAC-based therapeutic molecules. To develop BTK-targeting PROTAC degraders, a BTK-targeting arm was conjugated to a BTK-degradation arm by linkers with variable lengths (Fig. 1a, b, for details, please see Supplementary Information). As a result, the PROTAC molecules should bind both BTK and E3 ligase through the corresponding targeting arm and degradation arm, respectively. The drugs ibrutinib and spebrutinib were selected as the BTK-binding ligands, and pomalidomide (CRBN ligand) and RG-7112 (MDM2 ligand) were employed as corresponding E3 ligase binding partners. Based on our design principles (for details, please see Supplementary Information), a variety of BTK-targeting PROTAC molecules were prepared and evaluated. We found that CRBN-recruiting PROTACs were generally more effective than MDM2-recruiting ones. Among these CRBN-recruiting PROTACs, P13I with the conjugation of ibrutinib and pomalidomide demonstrated the best degrading ability (Fig. 1b). P13I induced 73% degradation of BTK at 10 nM and 89% at 100 nM in human Burkitt’s lymphoma, RAMOS cells. As a further validation, we examined the kinetics of P13I-induced BTK degradation in human ABC-DLBCL, HBL-1 cells. Western blot analysis showed that BTK degradation began at roughly 4 h, and was completed by around 24 h (Fig. 1c). With P13I treatment, halflives of BTK and C481S BTK were 4 h and 3 h, respectively. The results demonstrated that P13I could accelerate BTK degradation (for details, please see SI). Moreover, P13I could also efficiently degrade BTK in other NHL cell lines including MCL (Mino cells) and MM cell lines with a DC50 (50% protein degradation concentration) of 9.2 nM and 11.4 nM, respectively (Fig. 1d). Control experiments clearly demonstrated that ibrutinib, pomalidomide or unconjugated PROTAC arms (TA and DA) could not induce degradation of BTK (Fig. 1e). In contrast, ibrutinib or pomalidomide could competitively inhibit the degradation effect of P13I. Additionally, MG-132, a proteasome inhibitor, could completely disable the PROTAC effect. The same competitive

Single-cell RNA-seq reveals the diversity of trophoblast subtypes and patterns of differentiation in the human placenta

Abstract: The placenta is crucial for a successful pregnancy and the health of both the fetus and the pregnant woman. However, how the human trophoblast lineage is regulated, including the categorization of the placental cell subtypes is poorly understood. Here we performed single-cell RNA sequencing (RNA-seq) on sorted placental cells from first- and second-trimester human placentas. New subtypes of cells of the known cytotrophoblast cells (CTBs), extravillous trophoblast cells (EVTs), Hofbauer cells, and mesenchymal stromal cells were identified and cell-type-specific gene signatures were defined. Functionally, this study revealed many previously unknown functions of the human placenta. Notably, 102 polypeptide hormone genes were found to be expressed by various subtypes of placental cells, which suggests a complex and significant role of these hormones in regulating fetal growth and adaptations of maternal physiology to pregnancy. These results document human placental trophoblast differentiation at single-cell resolution and thus advance our understanding of human placentation during the early stage of pregnancy.

Suppression of m6A reader Ythdf2 promotes hematopoietic stem cell expansion

Abstract: Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2′s key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Spatial transcriptomic survey of human embryonic cerebral cortex by single-cell RNA-seq analysis.

Abstract: The cellular complexity of human brain development has been intensively investigated, although a regional characterization of the entire human cerebral cortex based on single-cell transcriptome analysis has not been reported. Here, we performed RNA-seq on over 4,000 individual cells from 22 brain regions of human mid-gestation embryos. We identified 29 cell sub-clusters, which showed different proportions in each region and the pons showed especially high percentage of astrocytes. Embryonic neurons were not as diverse as adult neurons, although they possessed important features of their destinies in adults. Neuron development was unsynchronized in the cerebral cortex, as dorsal regions appeared to be more mature than ventral regions at this stage. Region-specific genes were comprehensively identified in each neuronal sub-cluster, and a large proportion of these genes were neural disease related. Our results present a systematic landscape of the regionalized gene expression and neuron maturation of the human cerebral cortex.

A reappraisal of CTLA-4 checkpoint blockade in cancer immunotherapy

Abstract: It is assumed that anti-CTLA-4 antibodies cause tumor rejection by blocking negative signaling from B7-CTLA-4 interactions. Surprisingly, at concentrations considerably higher than plasma levels achieved by clinically effective dosing, the anti-CTLA-4 antibody Ipilimumab blocks neither B7 trans-endocytosis by CTLA-4 nor CTLA-4 binding to immobilized or cell-associated B7. Consequently, Ipilimumab does not increase B7 on dendritic cells (DCs) from either CTLA4 gene humanized (Ctla4h/h) or human CD34+ stem cell-reconstituted NSG™ mice. In Ctla4h/m mice expressing both human and mouse CTLA4 genes, anti-CTLA-4 antibodies that bind to human but not mouse CTLA-4 efficiently induce Treg depletion and Fc receptor-dependent tumor rejection. The blocking antibody L3D10 is comparable to the non-blocking Ipilimumab in causing tumor rejection. Remarkably, L3D10 progenies that lose blocking activity during humanization remain fully competent in inducing Treg depletion and tumor rejection. Anti-B7 antibodies that effectively block CD4 T cell activation and de novo CD8 T cell priming in lymphoid organs do not negatively affect the immunotherapeutic effect of Ipilimumab. Thus, clinically effective anti-CTLA-4 mAb causes tumor rejection by mechanisms that are independent of checkpoint blockade but dependent on the host Fc receptor. Our data call for a reappraisal of the CTLA-4 checkpoint blockade hypothesis and provide new insights for the next generation of safe and effective anti-CTLA-4 mAbs.

Circular RNA F-circEA produced from EML4-ALK fusion gene as a novel liquid biopsy biomarker for non-small cell lung cancer

Abstract: Dear Editor, Non-small cell lung cancer (NSCLC) accounts for over 75% cases of lung cancer, the leading cause of cancer deaths in the world. Most NSCLC patients are diagnosed at an advanced stage due to the inadequate screening program and late onset of clinical symptoms, leading to poor prognosis. Discovery of accurate and sensitive biomarkers is in urgent need. Recently, circular RNAs (circRNAs) are emerging as a novel biomarker because of their conservation, abundance, cell type-specific and tissue-specific expression, and their roles in disease progression. Increasing studies focus on molecular mechanisms of NSCLC with fusion genes, a hybrid of two otherwise-separated genes caused from aberrant chromosomal translocations. The fusion gene Echinoderm Microtubule-associated protein-Like 4 (EML4)Anaplastic Lymphoma Kinase (ALK) is present in 4% to 5% of NSCLC cases and generates oncogenic activity by activating ALK kinase. Recently, mounting evidence demonstrates that fusion genes not only encode fusion proteins involved in tumorigenesis, but also generate non-coding RNAs contributing to tumor progression. For example, the circRNA generated by the MLL/AF9 fusion gene (fcircM9) in leukemia exerts pro-proliferative and pro-oncogenic activities, prompting us to investigate whether the EML4-ALK fusion gene produces circRNA with clinical relevance in NSCLC. Unlike linear RNAs, circRNAs have a circular covalently-bonded structure, which endows circRNAs with higher tolerance to exonuclease digestion and prolonged lifetime in systemic circulation. CircRNAs are demonstrated to be enriched and stable in exosomes of peripheral blood. Compared to traditional biopsy biomarkers in tumor tissues, circRNAs in body fluids could be used as more convenient and non-invasive “liquid biopsy” biomarkers to detect tumor at early and late stages. Here, we report a novel circRNA named as F-circEA generated from the EML4-ALK fusion gene by back-splicing and identify its role in promoting tumor development. Notably, the existence of F-circEA in plasma suggests that F-circEA is a potentially novel “liquid biopsy” biomarker for diagnosis of EML4-ALK-positive NSCLC, guiding targeted therapy in clinic. We firstly investigated the existence of endogenous F-circEA in H2228 cells harboring the EML4-ALK variant 3b translocation. The presence of the EML4-ALK fusion gene in H2228 cells was verified by sequencing the reverse transcription PCR (RT-PCR) products amplified with F1/R1 primers (Fig. 1a, b). Total RNAs were extracted from H2228 or H1299 cells (negative control without the fusion gene), and subjected to RNase-R digestion to remove linear RNA molecules. By using F2/R2 primers (Fig. 1a), potential circRNAs produced by the EML4-ALK fusion gene were specifically found in H2228 samples (Fig. 1c, left), and the one with an apparent length of ~0.55 kb was Sanger sequenced to confirm the back-splice junction between the 5′ head of EML4 exon 4 fragment and 3′ tail of ALK exon 22 fragment (Fig. 1c, right), different from that in H3321 cells with the EML4-ALK variant 1 translocation. Moreover, F-circEA was further confirmed by RNA hybridization assays with or without RNase-R digestion using Plabeled probes crossing the junction site and fusion site, respectively (Fig. 1d; Supplementary Information, Figure S1a). The back-splice junction does not fit the GT-AG pattern of a U2 intron and there is no obvious complementary structure in the adjacent introns, suggesting that F-circEA might be produced through a mechanism distinct from the canonical back-splicing pathway. We speculate the back-splice site might be an unconventional U12 intron, however, the exact mechanism needs experimental investigation. The subcellular fractionation and RTqPCR analyses showed that F-circEA was mainly located in the cytoplasm of H2228 cells (Fig. 1e). As the EML4-ALK translocation is an oncogenic driver mutation associated with tumor proliferation, migration, and invasion, we planned to investigate the potential role of F-circEA in tumorigenesis. To this end, small interfering RNAs (siRNAs) targeting its backsplice junction were designed (Fig. 1a) and effectively knocked down the expression of endogenous F-circEA with minor influence on EML4-ALK mRNA level (Fig. 1f; Supplementary Information, Figure S1b). Transwell assays showed that F-circEA knockdown decreased cell migratory and invasion ability (Fig. 1g; Supplementary Information, Figure S1c), whereas it had little effect on cell proliferation and colony formation (Supplementary Information, Figure S1d and e). Moreover, these siRNAs had little effect on cell migration and invasion in H1299 cells without such fusion gene (Supplementary Information, Fig. S1f). To confirm these observations, the F-circEA expressing vector was constructed by cloning the circularizing sequence into the vector we made with reverse repeat of cirR-7 exons together with up-stream and down-stream flanking introns, which favored the formation of circular RNA (Fig. 1h). F-circEA was successfully expressed and correctly circularized in H1299 cells (Fig. 1i; Supplementary Information, Figure S1g and h), and was also predominantly located in the cytoplasm of H1299 and A549 cells (Fig. 1j; Supplementary Information, Figure S1i) that did not harbor the EML4-ALK translocation and therefore had no expression of endogenous fusion protein and corresponding circRNAs. Through Transwell assays and wound healing experiments, the cells expressing FcircEAs displayed higher migration and invasion ability than the cells expressing the empty vector in both H1299 (Fig. 1k; Supplementary Information, Figure S1j and k) and A549 cells (Supplementary Information, Figure S1l and m). Furthermore, the increased cell migration and invasion in F-circEA-expressing cells were attenuated upon the F-circEA silencing by siRNAs (Fig. 1l, m,

Structure of the intact 14-subunit human cytochrome c oxidase.

Abstract: Respiration is one of the most basic features of living organisms, and the electron transport chain complexes are probably the most complicated protein system in mitochondria. Complex-IV is the terminal enzyme of the electron transport chain, existing either as randomly scattered complexes or as a component of supercomplexes. NDUFA4 was previously assumed as a subunit of complex-I, but recent biochemical data suggested it may be a subunit of complex-IV. However, no structural evidence supporting this notion was available till now. Here we obtained the 3.3 A resolution structure of complex-IV derived from the human supercomplex I1III2IV1 and assigned the NDUFA4 subunit into complex-IV. Intriguingly, NDUFA4 lies exactly at the dimeric interface observed in previously reported crystal structures of complex-IV homodimer which would preclude complex-IV dimerization. Combining previous structural and biochemical data shown by us and other groups, we propose that the intact complex-IV is a monomer containing 14 subunits.

Structure of the human activated spliceosome in three conformational states.

Abstract: During each cycle of pre-mRNA splicing, the pre-catalytic spliceosome (B complex) is converted into the activated spliceosome (Bact complex), which has a well-formed active site but cannot proceed to the branching reaction. Here, we present the cryo-EM structure of the human Bact complex in three distinct conformational states. The EM map allows atomic modeling of nearly all protein components of the U2 small nuclear ribonucleoprotein (snRNP), including three of the SF3a complex and seven of the SF3b complex. The structure of the human Bact complex contains 52 proteins, U2, U5, and U6 small nuclear RNA (snRNA), and a pre-mRNA. Three distinct conformations have been captured, representing the early, mature, and late states of the human Bact complex. These complexes differ in the orientation of the Switch loop of Prp8, the splicing factors RNF113A and NY-CO-10, and most components of the NineTeen complex (NTC) and the NTC-related complex. Analysis of these three complexes and comparison with the B and C complexes reveal an ordered flux of components in the B-to-Bact and the Bact-to-B* transitions, which ultimately prime the active site for the branching reaction.

Circulating apoptotic bodies maintain mesenchymal stem cell homeostasis and ameliorate osteopenia via transferring multiple cellular factors.

Abstract: In the human body, 50–70 billion cells die every day, resulting in the generation of a large number of apoptotic bodies. However, the detailed biological role of apoptotic bodies in regulating tissue homeostasis remains unclear. In this study, we used Fas-deficient MRL/lpr and Caspase 3−/− mice to show that reduction of apoptotic body formation significantly impaired the self-renewal and osteo-/adipo-genic differentiation of bone marrow mesenchymal stem cells (MSCs). Systemic infusion of exogenous apoptotic bodies rescued the MSC impairment and also ameliorated the osteopenia phenotype in MRL/lpr, Caspase 3−/− and ovariectomized (OVX) mice. Mechanistically, we showed that MSCs were able to engulf apoptotic bodies via integrin αvβ3 and reuse apoptotic body-derived ubiquitin ligase RNF146 and miR-328-3p to inhibit Axin1 and thereby activate the Wnt/β-catenin pathway. Moreover, we used a parabiosis mouse model to reveal that apoptotic bodies participated in the circulation to regulate distant MSCs. This study identifies a previously unknown role of apoptotic bodies in maintaining MSC and bone homeostasis in both physiological and pathological contexts and implies the potential use of apoptotic bodies to treat osteoporosis.

Structure of the receptor-activated human TRPC6 and TRPC3 ion channels

Abstract: TRPC6 and TRPC3 are receptor-activated nonselective cation channels that belong to the family of canonical transient receptor potential (TRPC) channels. They are activated by diacylglycerol, a lipid second messenger. TRPC6 and TRPC3 are involved in many physiological processes and implicated in human genetic diseases. Here we present the structure of human TRPC6 homotetramer in complex with a newly identified high-affinity inhibitor BTDM solved by single-particle cryo-electron microscopy to 3.8 A resolution. We also present the structure of human TRPC3 at 4.4 A resolution. These structures show two-layer architectures in which the bell-shaped cytosolic layer holds the transmembrane layer. Extensive inter-subunit interactions of cytosolic domains, including the N-terminal ankyrin repeats and the C-terminal coiled-coil, contribute to the tetramer assembly. The high-affinity inhibitor BTDM wedges between the S5-S6 pore domain and voltage sensor-like domain to inhibit channel opening. Our structures uncover the molecular architecture of TRPC channels and provide a structural basis for understanding the mechanism of these channels.

Histone H3 lysine 4 monomethylation modulates long-range chromatin interactions at enhancers.

Abstract: Long-range chromatin interactions between enhancers and promoters are essential for transcription of many developmentally controlled genes in mammals and other metazoans. Currently, the exact mechanisms that connect distal enhancers to their specific target promoters remain to be fully elucidated. Here, we show that the enhancer-specific histone H3 lysine 4 monomethylation (H3K4me1) and the histone methyltransferases MLL3 and MLL4 (MLL3/4) play an active role in this process. We demonstrate that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of this histone modification leads to reduced levels of chromatin interactions and defects in gene activation during differentiation. H3K4me1 facilitates recruitment of the Cohesin complex, a known regulator of chromatin organization, to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin interactions between enhancers and promoters. Taken together, our results support a role for MLL3/4-dependent H3K4me1 in orchestrating long-range chromatin interactions at enhancers in mammalian cells.

A dynamic N 6 -methyladenosine methylome regulates intrinsic and acquired resistance to tyrosine kinase inhibitors.

Abstract: N6-methyladenosine (m6A) on mRNAs is critical for various biological processes, yet whether m6A regulates drug resistance remains unknown. Here we show that developing resistant phenotypes during tyrosine kinase inhibitor (TKI) therapy depends on m6A reduction resulting from FTO overexpression in leukemia cells. This deregulated FTO-m6A axis pre-exists in naive cell populations that are genetically homogeneous and is inducible/reversible in response to TKI treatment. Cells with mRNA m6A hypomethylation and FTO upregulation demonstrate more TKI tolerance and higher growth rates in mice. Either genetic or pharmacological restoration of m6A methylation through FTO deactivation renders resistant cells sensitive to TKIs. Mechanistically, the FTO-dependent m6A demethylation enhances mRNA stability of proliferation/survival transcripts bearing m6A and subsequently leads to increased protein synthesis. Our findings identify a novel function for the m6A methylation in regulating cell fate decision and demonstrate that dynamic m6A methylome is an additional epigenetic driver of reversible TKI-tolerance state, providing a mechanistic paradigm for drug resistance in cancer.

Skeletal muscle mitochondrial remodeling in exercise and diseases

Abstract: Skeletal muscle fitness and plasticity is an important determinant of human health and disease. Mitochondria are essential for maintaining skeletal muscle energy homeostasis by adaptive re-programming to meet the demands imposed by a myriad of physiologic or pathophysiological stresses. Skeletal muscle mitochondrial dysfunction has been implicated in the pathogenesis of many diseases, including muscular dystrophy, atrophy, type 2 diabetes, and aging-related sarcopenia. Notably, exercise counteracts the effects of many chronic diseases on skeletal muscle mitochondrial function. Recent studies have revealed a finely tuned regulatory network that orchestrates skeletal muscle mitochondrial biogenesis and function in response to exercise and in disease states. In addition, increasing evidence suggests that mitochondria also serve to “communicate” with the nucleus and mediate adaptive genomic re-programming. Here we review the current state of knowledge relevant to the dynamic remodeling of skeletal muscle mitochondria in response to exercise and in disease states.

METTL3-mediated N6-methyladenosine mRNA modification enhances long-term memory consolidation.

Abstract: The formation of long-term memory is critical for learning ability and social behaviors of humans and animals, yet its underlying mechanisms are largely unknown. We found that the efficacy of hippocampus-dependent memory consolidation is regulated by METTL3, an RNA N6-methyladenosine (m6A) methyltransferase, through promoting the translation of neuronal early-response genes. Such effect is exquisitely dependent on the m6A methyltransferase function of METTL3. Depleting METTL3 in mouse hippocampus reduces memory consolidation ability, yet unimpaired learning outcomes can be achieved if adequate training was given or the m6A methyltransferase function of METTL3 was restored. The abundance of METTL3 in wild-type mouse hippocampus is positively correlated with learning efficacy, and overexpression of METTL3 significantly enhances long-term memory consolidation. These findings uncover a direct role of RNA m6A modification in regulating long-term memory formation, and also indicate that memory efficacy difference among individuals could be compensated by repeated learning.

Malate transported from chloroplast to mitochondrion triggers production of ROS and PCD in Arabidopsis thaliana.

Abstract: Programmed cell death (PCD) is a fundamental biological process. Deficiency in MOSAIC DEATH 1 (MOD1), a plastid-localized enoyl-ACP reductase, leads to the accumulation of reactive oxygen species (ROS) and PCD, which can be suppressed by mitochondrial complex I mutations, indicating a signal from chloroplasts to mitochondria. However, this signal remains to be elucidated. In this study, through cloning and analyzing a series of mod1 suppressors, we reveal a comprehensive organelle communication pathway that regulates the generation of mitochondrial ROS and triggers PCD. We show that mutations in PLASTIDIAL NAD-DEPENDENT MALATE DEHYDROGENASE (plNAD-MDH), chloroplastic DICARBOXYLATE TRANSPORTER 1 (DiT1) and MITOCHONDRIAL MALATE DEHYDROGENASE 1 (mMDH1) can each rescue the ROS accumulation and PCD phenotypes in mod1, demonstrating a direct communication from chloroplasts to mitochondria via the malate shuttle. Further studies demonstrate that these elements play critical roles in the redox homeostasis and plant growth under different photoperiod conditions. Moreover, we reveal that the ROS level and PCD are significantly increased in malate-treated HeLa cells, which can be dramatically attenuated by knockdown of the human gene MDH2, an ortholog of Arabidopsis mMDH1. These results uncover a conserved malate-induced PCD pathway in plant and animal systems, revolutionizing our understanding of the communication between organelles.