Structural basis for double-stranded RNA recognition by SID1.

The nucleic acid transport properties of the systemic RNAi-defective (SID) 1 family make them attractive targets for developing RNA-based therapeutics and drugs. However, the molecular basis for double-stranded (ds) RNA recognition by SID1 family remains elusive. Here, we report the cryo-EM structures of Caenorhabditis elegans (c) SID1 alone and in complex with dsRNA, both at a resolution of 2.2 Å. The dimeric cSID1 interacts with two dsRNA molecules simultaneously. The dsRNA is located at the interface between ß-strand rich domain (BRD)1 and BRD2 and nearly parallel to the membrane plane. In addition to extensive ionic interactions between basic residues and phosphate backbone, several hydrogen bonds are formed between 2'-hydroxyl group of dsRNA and the contact residues. Additionally, the electrostatic potential surface shows three basic regions are fitted perfectly into three major grooves of dsRNA. These structural characteristics enable cSID1 to bind dsRNA in a sequence-independent manner and to distinguish between DNA and RNA. The cSID1 exhibits no conformational changes upon binding dsRNA, with the exception of a few binding surfaces. Structural mapping of dozens of loss-of-function mutations allows potential interpretation of their diverse functional mechanisms. Our study marks an important step toward mechanistic understanding of the SID1 family-mediated dsRNA uptake.

Structural insight into the human SID1 transmembrane family member 2 reveals its lipid hydrolytic activity.

The systemic RNAi-defective (SID) transmembrane family member 2 (SIDT2) is a putative nucleic acid channel or transporter that plays essential roles in nucleic acid transport and lipid metabolism. Here, we report the cryo-electron microscopy (EM) structures of human SIDT2, which forms a tightly packed dimer with extensive interactions mediated by two previously uncharacterized extracellular/luminal ß-strand-rich domains and the unique transmembrane domain (TMD). The TMD of each SIDT2 protomer contains eleven transmembrane helices (TMs), and no discernible nucleic acid conduction pathway has been identified within the TMD, suggesting that it may act as a transporter. Intriguingly, TM3-6 and TM9-11 form a large cavity with a putative catalytic zinc atom coordinated by three conserved histidine residues and one aspartate residue lying approximately 6 Å from the extracellular/luminal surface of the membrane. Notably, SIDT2 can hydrolyze C18 ceramide into sphingosine and fatty acid with a slow rate. The information presented advances the understanding of the structure-function relationships in the SID1 family proteins.

Whole exome sequencing in unexplained recurrent miscarriage families identified novel pathogenic genetic causes of euploid miscarriage.

STUDY QUESTION: Can whole exome sequencing (WES) followed by trio bioinformatics analysis identify novel pathogenic genetic causes of first trimester euploid miscarriage? SUMMARY ANSWER: We identified genetic variants in six candidate genes that indicated plausible underlying causes of first-trimester euploid miscarriage. WHAT IS KNOWN ALREADY: Previous studies have identified several monogenic causes of Mendelian inheritance in euploid miscarriages. However, most of these studies are without trio analyses and lack cellular and animal models to validate the functional effect of putative pathogenic variants. STUDY DESIGN, SIZE, DURATION: Eight unexplained recurrent miscarriage (URM) couples and corresponding euploid miscarriages were included in our study for whole genome sequencing (WGS) and WES followed by trio bioinformatics analysis. Knock-in mice with Rry2 and Plxnb2 variants and immortalized human trophoblasts were utilized for functional study. Additional 113 unexplained miscarriages were included to identify the mutation prevalence of specific genes by multiplex PCR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole blood from URM couples and their <13 weeks gestation miscarriage products were both collected for WES, and all variants in selected genes were verified by Sanger sequencing. Different stage C57BL/6J wild-type mouse embryos were collected for immunofluorescence. Ryr2N1552S/+, Ryr2R137W/+, Plxnb2D1577E/+, and Plxnb2R465Q/+ point mutation mice were generated and backcrossed. Matrigel-coated transwell invasion assays and wound-healing assays were performed using HTR-8/SVneo cells transfected with PLXNB2 small-interfering RNA and negative control. Multiplex PCR was performed focusing on RYR2 and PLXNB2. MAIN RESULTS AND THE ROLE OF CHANCE: Six novel candidate genes, including ATP2A2, NAP1L1, RYR2, NRK, PLXNB2, and SSPO, were identified. Immunofluorescence staining showed that ATP2A2, NAP1L1, RyR2, and PLXNB2 were widely expressed from the zygote to the blastocyst stage in mouse embryos. Although compound heterozygous mice with Rry2 and Plxnb2 variants did not show embryonic lethality, the number of pups per litter was significantly reduced when backcrossing Ryr2N1552S/+ ♂ with Ryr2R137W/+ ♀ or Plxnb2D1577E/+ ♂ with Plxnb2R465Q/+ ♀ (P < 0.05), which were in accordance with the sequencing results of Family 2 and Family 3, and the proportion of Ryr2N1552S/+ offspring was significantly lower when Ryr2N1552S/+ female mice were backcrossed with Ryr2R137W/+ male mice (P < 0.05). Moreover, siRNA-mediated PLXNB2 knockdown inhibited the migratory and invasive abilities of immortalized human trophoblasts. Besides, additional 10 variants of RYR2 and PLXNB2 were detected in 113 unexplained euploid miscarriages by multiplex PCR. LIMITATIONS, REASONS FOR CAUTION: The relatively small number of samples is a limitation of our study which may result in the identification of variants in unique candidate genes with no definitive although plausible causal effect. Larger cohorts are needed to replicate these findings and additional functional research is needed to confirm the pathogenic effects of these variants. Moreover, the sequencing coverage restricted the detection of low-level parental mosaic variants. WIDER IMPLICATIONS OF THE FINDINGS: For first-trimester euploid miscarriage, variants in unique genes may be underlying genetic etiologies and WES on trio could be an ideal model to identify potential genetic causes, which could facilitate individualized precise diagnostic and therapeutic regimens in the future. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the National Key Research and Development Program of China (2021YFC2700604), National Natural Science Foundation of China (31900492, 82101784, 82171648), Basic Science Center Program of the National Natural Science Foundation of China (31988101), Key Research and Development Program of Shandong Province (2021LCZX02), Natural Science Foundation of Shandong Province (ZR2020QH051), Natural Science Foundation of Jiangsu Province (BK20200223), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201812154) and Young Scholars Program of Shandong University. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Structure of the human Meckel-Gruber protein Meckelin.

Mutations in the Meckelin gene account for most cases of the Meckel-Gruber syndrome, the most severe ciliopathy with a 100% mortality rate. Here, we report a 3.3-Å cryo­electron microscopy structure of human Meckelin (also known as TMEM67 and MKS3). The structure reveals a unique protein fold consisting of an unusual cysteine-rich domain that folds as an arch bridge stabilized by 11 pairs of disulfide bonds, a previously uncharacterized domain named ß sheet­rich domain, a previously unidentified seven-transmembrane fold wherein TM4 to TM6 are broken near the cytoplasmic surface of the membrane, and a coiled-coil domain placed below the transmembrane domain. Meckelin forms a stable homodimer with an extensive dimer interface. Our structure establishes a framework for dissecting the function and disease mechanisms of Meckelin.

Structural Basis for the Modulation of Ryanodine Receptors.

Historically, ryanodine receptors (RyRs) have presented unique challenges for high-resolution structural determination despite long-standing interest in their role in excitation-contraction coupling. Owing to their large size (nearly 2.2 MDa), high-resolution structures remained elusive until the advent of cryogenic electron microscopy (cryo-EM) techniques. In recent years, structures for both RyR1 and RyR2 have been solved at near-atomic resolution. Furthermore, recent reports have delved into their more complex structural associations with key modulators - proteins such as the dihydropyridine receptor (DHPR), FKBP12/12.6, and calmodulin (CaM), as well as ions and small molecules including Ca2+, ATP, caffeine, and PCB95. This review addresses the modulation of RyR1 and RyR2, in addition to the impact of such discoveries on intracellular Ca2+ dynamics and biophysical properties.

Molecular basis for allosteric regulation of the type 2 ryanodine receptor channel gating by key modulators.

The type 2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report 4 cryo-electron microscopy (cryo-EM) structures of porcine RyR2 bound to distinct modulators that, together with our published structures, provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the central domain that facilitates the dilation of the S6 bundle but is insufficient to open the pore. The small-molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state even in the presence of Ca2+ and PCB95. Although the channel is open when PCB95 is replaced by caffeine and adenosine 5'-triphosphate (ATP), neither of the modulators alone can sufficiently counter the antagonistic effect to open the channel. Our study marks an important step toward mechanistic understanding of the sophisticated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.

TRIM66 reads unmodified H3R2K4 and H3K56ac to respond to DNA damage in embryonic stem cells.

Recognition of specific chromatin modifications by distinct structural domains within "reader" proteins plays a critical role in the maintenance of genomic stability. However, the specific mechanisms involved in this process remain unclear. Here we report that the PHD-Bromo tandem domain of tripartite motif-containing 66 (TRIM66) recognizes the unmodified H3R2-H3K4 and acetylated H3K56. The aberrant deletion of Trim66 results in severe DNA damage and genomic instability in embryonic stem cells (ESCs). Moreover, we find that the recognition of histone modification by TRIM66 is critical for DNA damage repair (DDR) in ESCs. TRIM66 recruits Sirt6 to deacetylate H3K56ac, negatively regulating the level of H3K56ac and facilitating the initiation of DDR. Importantly, Trim66-deficient blastocysts also exhibit higher levels of H3K56ac and DNA damage. Collectively, the present findings indicate the vital role of TRIM66 in DDR in ESCs, establishing the relationship between histone readers and maintenance of genomic stability.

Modulation of cardiac ryanodine receptor 2 by calmodulin.

The high-conductance intracellular calcium (Ca2+) channel RyR2 is essential for the coupling of excitation and contraction in cardiac muscle. Among various modulators, calmodulin (CaM) regulates RyR2 in a Ca2+-dependent manner. Here we reveal the regulatory mechanism by which porcine RyR2 is modulated by human CaM through the structural determination of RyR2 under eight conditions. Apo-CaM and Ca2+-CaM bind to distinct but overlapping sites in an elongated cleft formed by the handle, helical and central domains. The shift in CaM-binding sites on RyR2 is controlled by Ca2+ binding to CaM, rather than to RyR2. Ca2+-CaM induces rotations and intradomain shifts of individual central domains, resulting in pore closure of the PCB95 and Ca2+-activated channel. By contrast, the pore of the ATP, caffeine and Ca2+-activated channel remains open in the presence of Ca2+-CaM, which suggests that Ca2+-CaM is one of the many competing modulators of RyR2 gating.

Structure of the human plasma membrane Ca2+-ATPase 1 in complex with its obligatory subunit neuroplastin.

Plasma membrane Ca2+-ATPases (PMCAs) are key regulators of global Ca2+ homeostasis and local intracellular Ca2+ dynamics. Recently, Neuroplastin (NPTN) and basigin were identified as previously unrecognized obligatory subunits of PMCAs that dramatically increase the efficiency of PMCA-mediated Ca2+ clearance. Here, we report the cryo-EM structure of human PMCA1 (hPMCA1) in complex with NPTN at a resolution of 4.1 Å for the overall structure and 3.9 Å for the transmembrane domain. The single transmembrane helix of NPTN interacts with the TM8-9-linker and TM10 of hPMCA1. The subunits are required for the hPMCA1 functional activity. The NPTN-bound hPMCA1 closely resembles the E1-Mg2+ structure of endo(sarco)plasmic reticulum Ca2+ ATPase and the Ca2+ site is exposed through a large open cytoplasmic pathway. This structure provides insight into how the subunits bind to the PMCAs and serves as an important basis for understanding the functional mechanisms of this essential calcium pump family.

Crystal structure of triple-BRCT-domain of ECT2 and insights into the binding characteristics to CYK-4.

Homo sapiens ECT2 is a cell cycle regulator that plays critical roles in cytokinesis. ECT2 activity is restrained during interphase via intra-molecular interactions that involve its N-terminal triple-BRCT-domain and its C-terminal DH-PH domain. At anaphase, this self-inhibitory mechanism is relieved by Plk1-phosphorylated CYK-4, which directly engages the ECT2 BRCT domain. To provide a structural perspective for this auto-inhibitory property, we solved the crystal structure of the ECT2 triple-BRCT-domain. In addition, we systematically analyzed the interaction between the ECT2 BRCT domains with phospho-peptides derived from its binding partner CYK-4, and have identified Ser164 as the major phospho-residue that links CYK-4 to the second ECT2 BRCT domain.

Crystal structure of tRNA m1G9 methyltransferase Trm10: insight into the catalytic mechanism and recognition of tRNA substrate.

Transfer RNA (tRNA) methylation is necessary for the proper biological function of tRNA. The N(1) methylation of guanine at Position 9 (m(1)G9) of tRNA, which is widely identified in eukaryotes and archaea, was found to be catalyzed by the Trm10 family of methyltransferases (MTases). Here, we report the first crystal structures of the tRNA MTase spTrm10 from Schizosaccharomyces pombe in the presence and absence of its methyl donor product S-adenosyl-homocysteine (SAH) and its ortholog scTrm10 from Saccharomyces cerevisiae in complex with SAH. Our crystal structures indicated that the MTase domain (the catalytic domain) of the Trm10 family displays a typical SpoU-TrmD (SPOUT) fold. Furthermore, small angle X-ray scattering analysis reveals that Trm10 behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. We also performed tRNA MTase assays and isothermal titration calorimetry experiments to investigate the catalytic mechanism of Trm10 in vitro. In combination with mutational analysis and electrophoretic mobility shift assays, our results provide insights into the substrate tRNA recognition mechanism of Trm10 family MTases.

Crystal structure and functional characterization of the human RBM25 PWI domain and its flanking basic region.

Human RBM25 (RNA-binding motif protein 25) is a novel splicing factor that contains a PWI domain, a newly identified RNA/DNA-binding domain, and regulates Bcl-x pre-mRNA alternative splicing. The flanking basic region has been suggested to serve as a co-operative partner of the PWI domain in the binding of nucleic acids, but the structure of this basic region is unknown. In the present paper, we report the crystal structure of the RBM25 PWI domain and its flanking basic region. The PWI domain is revealed to comprise a conserved four-helix bundle, and the flanking basic region forms two α-helices and associates with helix H4 of the PWI domain. These interactions promote directly the formation of an enlarged nucleic-acid-binding platform. Structure-guided mutagenesis reveals a positively charged nucleic-acid-binding surface in the RBM25 PWI domain that is entirely different from that in the SRm160 PWI domain. Furthermore, we show that the promotion of the pro-apoptotic Bcl-xS isoform expression by RBM25 is facilitated by the PWI domain in vivo. Thus the present study suggests that the PWI domain plays an important role in the regulation of Bcl-x pre-mRNA alternative splicing.

Specific recognition of phosphorylated tail of H2AX by the tandem BRCT domains of MCPH1 revealed by complex structure.

MCPH1 is especially important for linking chromatin remodeling to DNA damage response. It contains three BRCT (BRCA1-carboxyl terminal) domains. The N-terminal region directly binds with chromatin remodeling complex SWI-SNF, and the C-terminal BRCT2-BRCT3 domains (tandem BRCT domains) are involved in cellular DNA damage response. The MCPH1 gene associates with evolution of brain size, and its variation can cause primary microcephaly. In this study we solve the crystal structures of MCPH1 natural variant (A761) C-terminal tandem BRCT domains alone as well as in complex with γH2AX tail. Compared with other structures of tandem BRCT domains, the most significant differences lie in phosphopeptide binding pocket. Additionally, fluorescence polarization assays demonstrate that MCPH1 tandem BRCT domains show a binding selectivity on pSer +3 and prefer to bind phosphopeptide with free COOH-terminus. Taken together, our research provides new structural insights into BRCT-phosphopeptide recognition mechanism.