Molecular mechanism of specific HLA-A mRNA recognition by the RNA-binding-protein hMEX3B to promote tumor immune escape.

Immunotherapy, including immune checkpoint inhibitors and adoptive cell transfer, has obtained great progress, but their efficiencies vary among patients due to the genetic and epigenetic differences. Human MEX3B (hMEX3B) protein is an RNA-binding protein that contains two KH domains at the N-terminus and a RING domain at its C-terminus, which has the activity of E3 ubiquitin ligase and is essential for RNA degradation. Current evidence suggests that hMEX3B is involved in many important biological processes, including tumor immune evasion and HLA-A regulation, but the sequence of substrate RNA recognized by hMEX3B and the functional molecular mechanisms are unclear. Here, we first screened the optimized hMEX3B binding sequence on the HLA-A mRNA and reported that the two tandem KH domains can bind with their substrate one hundred times more than the individual KH domains. We systematically investigated the binding characteristics between the two KH domains and their RNA substrates by nuclear magnetic resonance (NMR). Based on this information and the small-angle X-ray scattering (SAXS) data, we used molecular dynamics simulations to obtain structural models of KH domains in complex with their corresponding RNAs. By analyzing the models, we noticed that on the KH domains' variable loops, there were two pairs of threonines and arginines that can disrupt the recognition of the RNA completely, and this influence had also been verified both in vitro and in vivo. Finally, we presented a functional model of the hMEX3B protein, which indicated that hMEX3B regulated the degradation of its substrate mRNAs in many biological processes. Taken together, our research illustrated how the hMEX3B protein played a key role in translation inhibition during the immune response to tumor cells and provided an idea and a lead for the study of the molecular mechanism and function of other MEX3 family proteins.

Structural and dynamic mechanisms for coupled folding and tRNA recognition of a translational T-box riboswitch.

T-box riboswitches are unique riboregulators where gene regulation is mediated through interactions between two highly structured RNAs. Despite extensive structural insights, how RNA-RNA interactions drive the folding and structural transitions of T-box to achieve functional conformations remains unclear. Here, by combining SAXS, single-molecule FRET and computational modeling, we elaborate the folding energy landscape of a translational T-box aptamer consisting of stems I, II and IIA/B, which Mg2+-induced global folding and tRNA binding are cooperatively coupled. smFRET measurements reveal that high Mg2+ stabilizes IIA/B and its stacking on II, which drives the pre-docking of I and II into a competent conformation, subsequent tRNA binding promotes docking of I and II to form a high-affinity tRNA binding groove, of which the essentiality of IIA/B and S-turn in II is substantiated with mutational analysis. We highlight a delicate balance among Mg2+, the intra- and intermolecular RNA-RNA interactions in modulating RNA folding and function.

A single amino acid substitution in the capsid protein of Zika virus contributes to a neurovirulent phenotype.

Increasing evidence shows the African lineage Zika virus (ZIKV) displays a more severe neurovirulence compared to the Asian ZIKV. However, viral determinants and the underlying mechanisms of enhanced virulence phenotype remain largely unknown. Herein, we identify a panel of amino acid substitutions that are unique to the African lineage of ZIKVs compared to the Asian lineage by phylogenetic analysis and sequence alignment. We then utilize reverse genetic technology to generate recombinant ZIKVs incorporating these lineage-specific substitutions based on an infectious cDNA clone of Asian ZIKV. Through in vitro characterization, we discover a mutant virus with a lysine to arginine substitution at position 101 of capsid (C) protein (termed K101R) displays a larger plaque phenotype, and replicates more efficiently in various cell lines. Moreover, K101R replicates more efficiently in mouse brains and induces stronger inflammatory responses than the wild type (WT) virus in neonatal mice. Finally, a combined analysis reveals the K101R substitution promotes the production of mature C protein without affecting its binding to viral RNA. Our study identifies the role of K101R substitution in the C protein in contributing to the enhanced virulent phenotype of the African lineage ZIKV, which expands our understanding of the complexity of ZIKV proteins.

A review on recent advances in methods for site-directed spin labeling of long RNAs.

RNAs are important biomolecules that play essential roles in various cellular processes and are crucially linked with many human diseases. The key to elucidate the mechanisms underlying their biological functions and develop RNA-based therapeutics is to investigate RNA structure and dynamics and their connections to function in detail using a variety of approaches. Magnetic resonance techniques including paramagnetic nuclear magnetic resonance (NMR) and electron magnetic resonance (EPR) spectroscopies have proved to be powerful tools to gain insights into such properties. The prerequisites for paramagnetic NMR and EPR studies on RNAs are to achieve site-specific spin labeling of the intrinsically diamagnetic RNAs, which however is not trivial, especially for long ones. In this review, we present some covalent labeling strategies that allow site-specific introduction of electron spins to long RNAs. Generally, these strategies include assembly of long RNAs via enzymatic ligation of short oligonucleotides, co- and post-transcriptional site-specific labeling empowered with the unnatural base pair system, and direct enzymatic functionalization of natural RNAs. We introduce a few case studies to discuss the advantages and limitations of each strategy, and to provide a vision for the future development.

Zika virus RNA structure controls its unique neurotropism by bipartite binding to Musashi-1.

Human RNA binding protein Musashi-1 (MSI1) plays a critical role in neural progenitor cells (NPCs) by binding to various host RNA transcripts. The canonical MSI1 binding site (MBS), A/GU(1-3)AG single-strand motif, is present in many RNA virus genomes, but only Zika virus (ZIKV) genome has been demonstrated to bind MSI1. Herein, we identified the AUAG motif and the AGAA tetraloop in the Xrn1-resistant RNA 2 (xrRNA2) as the canonical and non-canonical MBS, respectively, and both are crucial for ZIKV neurotropism. More importantly, the unique AGNN-type tetraloop is evolutionally conserved, and distinguishes ZIKV from other known viruses with putative MBSs. Integrated structural analysis showed that MSI1 binds to the AUAG motif and AGAA tetraloop of ZIKV in a bipartite fashion. Thus, our results not only identified an unusual viral RNA structure responsible for MSI recognition, but also revealed a role for the highly structured xrRNA in controlling viral neurotropism.

Structural insights into translation regulation by the THF-II riboswitch.

In bacteria, expression of folate-related genes is controlled by the tetrahydrofolate (THF) riboswitch in response to specific binding of THF and its derivatives. Recently, a second class of THF riboswitches, named THF-II, was identified in Gram-negative bacteria, which exhibit distinct architecture from the previously characterized THF-I riboswitches found in Gram-positive bacteria. Here, we present the crystal structures of the ligand-bound THF-II riboswitch from Mesorhizobium loti. These structures exhibit a long rod-like fold stabilized by continuous base pair and base triplet stacking across two helices of P1 and P2 and their interconnecting ligand-bound binding pocket. The pterin moiety of the ligand docks into the binding pocket by forming hydrogen bonds with two highly conserved pyrimidines in J12 and J21, which resembles the hydrogen-bonding pattern at the ligand-binding site FAPK in the THF-I riboswitch. Using small-angle X-ray scattering and isothermal titration calorimetry, we further characterized the riboswitch in solution and reveal that Mg2+ is essential for pre-organization of the binding pocket for efficient ligand binding. RNase H cleavage assay indicates that ligand binding reduces accessibility of the ribosome binding site in the right arm of P1, thus down-regulating the expression of downstream genes. Together, these results provide mechanistic insights into translation regulation by the THF-II riboswitch.

3D Structural Analysis of Long Noncoding RNA by Small Angle X-ray Scattering and Computational Modeling.

Small angle X-ray scattering (SAXS) has been widely applied as an enabling integrative technique for comprehensive analysis of the structure of biomacromolecules by multiple, complementary techniques in solution. SAXS in combination with computational modeling can be a powerful strategy bridging the secondary and 3D structural analysis of large RNAs, including the long noncoding RNAs (lncRNA). Here, we outline the major procedures and techniques in the combined use of SAXS and computational modeling for 3D structural characterization of a lncRNA, the subgenomic flaviviral RNA from Zika virus. lncRNA production and purification, RNA buffer and sample preparation for SAXS experiments, SAXS data collection and analysis, SAXS-aided RNA 3D structure prediction, and computational modeling are described.

Deriving RNA topological structure from SAXS.

Structures of well-folded RNA molecules can be determined with atomic resolution by either X-ray crystallography, cryo-EM, or NMR spectroscopy, but those of conformationally-flexible RNAs often are difficult to study with these methods. However, flexible RNAs have biological relevance and likely represent the majority of the RNA conformational space. Due to the high electron density of the phosphate-sugar backbone, RNA is very sensitive to small-angle X-ray scattering (SAXS), and SAXS data can be recorded with sub-µM concentrations and under near-physiological solution conditions without the need for labeling. For these reasons, SAXS has significant advantages over other techniques for obtaining global structural information of flexible RNAs in the form of molecular envelopes or low-resolution topological structural models. The SAXS-derived information is extremely valuable for bridging secondary structure data, often determined by other techniques, with a three-dimensional structure description. In this chapter, we present a detailed account of the principle, algorithms, and experimental and computational protocols for topological structure determination of RNA molecules in solution. To illustrate the applications of the methodology, we provide several case studies that cover a broad spectrum of the RNA conformational landscape.

Phosphorothioate-Based Site-Specific Labeling of Large RNAs for Structural and Dynamic Studies.

Pulsed electron-electron double resonance (PELDOR) spectroscopy, X-ray scattering interferometry (XSI), and single-molecule Förster resonance energy transfer (smFRET) are molecular rulers that provide inter- or intramolecular pair-wise distance distributions in the nanometer range, thus being ideally suitable for structural and dynamic studies of biomolecules including RNAs. The prerequisite for such applications requires site-specific labeling of biomolecules with spin labels, gold nanoparticles, and fluorescent tags, respectively. Recently, site-specific labeling of large RNAs has been achieved by a combination of transcription of an expanded genetic alphabet containing A-T/G-C base pairs and NaM-TPT3 unnatural base pair (UBP) with post-transcriptional modifications at UBP bases by click chemistry or amine-NHS ester reactions. However, due to the bulky sizes of functional groups or labeling probes used, such strategies might cause structural perturbation and decrease the accuracy of distance measurements. Here, we synthesize an α-thiophosphorylated variant of rTPT3TP (rTPT3αS), which allows for post-transcriptional site-specific labeling of large RNAs at the internal α-phosphate backbone via maleimide-modified probes. Subsequent PELDOR, XSI, and smFRET measurements result in narrower distance distributions than labeling at the TPT3 base. The presented strategy provides a new route to empower the molecular rulers for structural and dynamic studies of large RNA and its complex.

Recent advances in RNA structurome.

RNA structures are essential to support RNA functions and regulation in various biological processes. Recently, a range of novel technologies have been developed to decode genome-wide RNA structures and novel modes of functionality across a wide range of species. In this review, we summarize key strategies for probing the RNA structurome and discuss the pros and cons of representative technologies. In particular, these new technologies have been applied to dissect the structural landscape of the SARS-CoV-2 RNA genome. We also summarize the functionalities of RNA structures discovered in different regulatory layers-including RNA processing, transport, localization, and mRNA translation-across viruses, bacteria, animals, and plants. We review many versatile RNA structural elements in the context of different physiological and pathological processes (e.g., cell differentiation, stress response, and viral replication). Finally, we discuss future prospects for RNA structural studies to map the RNA structurome at higher resolution and at the single-molecule and single-cell level, and to decipher novel modes of RNA structures and functions for innovative applications.

Long-range distance determination in fully deuterated RNA with pulsed EPR spectroscopy.

Pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy is powerful in structure and dynamics study of biological macromolecules by providing distance distribution information ranging from 1.8 to 6 nm, providing that the biomolecules are site-specifically labeled with paramagnetic tags. However, long distances up to 16 nm have been measured on perdeuterated and spin-labeled proteins in deuterated solvent by PELDOR. Here we demonstrate long-range distance measurement on a large RNA, the 97-nucleotide 3'SL RNA element of the Dengue virus 2 genome, by combining a posttranscriptional site-directed spin labeling method using an unnatural basepair system with RNA perdeuteration by enzymatic synthesis using deuterated nucleotides. The perdeuteration removes the coupling of the electron spins of the nitroxide spin labels from the proton nuclear spin system of the RNA and does extend the observation time windows of PELDOR up to 50 µs. This enables one to determine long distances up to 14 nm for large RNAs and their conformational flexibility.

Pseudoknot length modulates the folding, conformational dynamics, and robustness of Xrn1 resistance of flaviviral xrRNAs.

To understand how RNA dynamics is regulated and connected to its function, we investigate the folding, conformational dynamics and robustness of Xrn1 resistance of a set of flaviviral xrRNAs using SAXS, smFRET and in vitro enzymatic assays. Flaviviral xrRNAs form discrete ring-like 3D structures, in which the length of a conserved long-range pseudoknot (PK2) ranges from 2 bp to 7 bp. We find that xrRNAs' folding, conformational dynamics and Xrn1 resistance are strongly correlated and highly Mg2+-dependent, furthermore, the Mg2+-dependence is modulated by PK2 length variations. xrRNAs with long PK2 require less Mg2+ to stabilize their folding, exhibit reduced conformational dynamics and strong Xrn1 resistance even at low Mg2+, and tolerate mutations at key tertiary motifs at high Mg2+, which generally are destructive to xrRNAs with short PK2. These results demonstrate an unusual regulatory mechanism of RNA dynamics providing insights into the functions and future biomedical applications of xrRNAs.

Structural mechanism for modulation of functional amyloid and biofilm formation by Staphylococcal Bap protein switch.

The Staphylococcal Bap proteins sense environmental signals (such as pH, [Ca2+ ]) to build amyloid scaffold biofilm matrices via unknown mechanisms. We here report the crystal structure of the aggregation-prone region of Staphylococcus aureus Bap which adopts a dumbbell-shaped fold. The middle module (MM) connecting the N-terminal and C-terminal lobes consists of a tandem of novel double-Ca2+ -binding motifs involved in cooperative interaction networks, which undergoes Ca2+ -dependent order-disorder conformational switches. The N-terminal lobe is sufficient to mediate amyloid aggregation through liquid-liquid phase separation and maturation, and subsequent biofilm formation under acidic conditions. Such processes are promoted by disordered MM at low [Ca2+ ] but inhibited by ordered MM stabilized by Ca2+ binding, with inhibition efficiency depending on structural integrity of the interaction networks. These studies illustrate a novel protein switch in pathogenic bacteria and provide insights into the mechanistic understanding of Bap proteins in modulation of functional amyloid and biofilm formation, which could be implemented in the anti-biofilm drug design.

A novel partially open state of SHP2 points to a "multiple gear" regulation mechanism.

The protein tyrosine phosphatase SHP2 mediates multiple signal transductions in various cellular pathways, controlled by a variety of upstream inputs. SHP2 dysregulation is causative of different types of cancers and developmental disorders, making it a promising drug target. However, how SHP2 is modulated by its different regulators remains largely unknown. Here, we use single-molecule fluorescence resonance energy transfer and molecular dynamics simulations to investigate this question. We identify a partially open, semiactive conformation of SHP2 that is intermediate between the known open and closed states. We further demonstrate a "multiple gear" regulatory mechanism, in which different activators (e.g., insulin receptor substrate-1 and CagA), oncogenic mutations (e.g., E76A), and allosteric inhibitors (e.g., SHP099) can shift the equilibrium of the three conformational states and regulate SHP2 activity to different levels. Our work reveals the essential role of the intermediate state in fine-tuning the activity of SHP2, which may provide new opportunities for drug development for relevant cancers.

[Life history traits of an Echinochloa crus-galli var. crus-galli population with extreme adaptations to rice planting systems]. / 特异性适应稻作系统的稗草种群生活史特性.

Long-term herbicide application may facilitate the adaptive evolution of weed populations. With Echinochloa crus-galli var. crus-galli population A from a rice field used for the experiment of effectiveness of herbicide in Nanling County, Anhui Province, we conducted common garden experi-ments with seeds of population A and three control populations collected from normal rice fields. Compared with the three control populations, population A had significantly lower seed production for individual plant, but higher 1000-seed weight. Population A had faster in seedling growth, higher in number of reproductive tillers, shorter life span, lower in height and biomass of individual plant, as well as lower in sensitivity to herbicide penoxsulam. Individuals from population A survived from 2× label dose (60 g·hm-2) of penoxsulam treatment at the three- to four-leaf stage showed significantly reduction in plant height, biomass, and mature seed production (1066 seeds per plant), but no difference in heading period, number of reproductive tillers, number of seeds per raceme and 1000-seed weight. The short lifespan, heavy seeds, dwarf architecture, more reproductive tillers and penoxsulam resistance made E. crus-galli var. crus-galli population A extremely adapting to rice planting systems, which should be prevented to spread to normal rice fields.

Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions.

Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs using an expanded genetic alphabet containing the NaM-TPT3 unnatural base pair (UBP). An alkyne-modified TPT3 ribonucleotide triphosphate (rTPT3COTP) is synthesized and site-specifically incorporated into large RNAs by in vitro transcription, which allows attachment of the azide-containing nitroxide through click chemistry. We validate this strategy by SDSL of a 419-nucleotide ribonuclease P (RNase P) RNA from Bacillus stearothermophilus under non-denaturing conditions. The effects of site-directed UBP incorporation and subsequent spin labeling on the global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, Sedimentation Velocity Analytical Ultracentrifugation and enzymatic assay. Continuous-Wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron-Electron Double Resonance measurements yield an inter-spin distance distribution that agrees with the crystal structure. The labeling strategy as presented overcomes the size constraint of RNA labeling, opening new avenues of spin labeling and EPR spectroscopy for investigating the structure and dynamics of large RNAs.

Molecular mechanisms underlying the extreme mechanical anisotropy of the flaviviral exoribonuclease-resistant RNAs (xrRNAs).

Mechanical anisotropy is an essential property for many biomolecules to assume their structures, functions and applications, however, the mechanisms for their direction-dependent mechanical responses remain elusive. Herein, by using a single-molecule nanopore sensing technique, we explore the mechanisms of directional mechanical stability of the xrRNA1 RNA from ZIKA virus (ZIKV), which forms a complex ring-like architecture. We reveal extreme mechanical anisotropy in ZIKV xrRNA1 which highly depends on Mg2+ and the key tertiary interactions. The absence of Mg2+ and disruption of the key tertiary interactions strongly affect the structural integrity and attenuate mechanical anisotropy. The significance of ring structures in RNA mechanical anisotropy is further supported by steered molecular dynamics simulations in combination with force distribution analysis. We anticipate the ring structures can be used as key elements to build RNA-based nanostructures with controllable mechanical anisotropy for biomaterial and biomedical applications.

Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription.

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3'SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3'SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.

The microtubule end-binding affinity of EB1 is enhanced by a dimeric organization that is susceptible to phosphorylation.

In cells, microtubule dynamics are regulated by plus-end tracking proteins (+TIPs). End-binding protein 1 (EB1, also known as MAPRE1) acts as a master regulator of +TIP networks by targeting the growing ends of microtubules and recruiting other factors. However, the molecular mechanism underlying high-affinity binding of EB1 to microtubule ends remains an open area of research. Using single-molecule imaging, we show that the end-binding kinetics of EB1 change when the polymerization and hydrolysis rates of tubulin dimers are altered, confirming that EB1 binds to GTP-tubulin and/or GDP-Pi-tubulin at microtubule growing ends. The affinity of wild-type EB1 to these sites is higher than that of monomeric EB1 mutants, suggesting that both calponin homology domains present in the EB1 dimer contribute to end binding. Introduction of phosphomimetic mutations into the EB1 linker domain weakens the end-binding affinity and confers a more curved conformation on the EB1 dimer without compromising dimerization, suggesting that the overall architecture of EB1 is important for its end-binding affinity. Taken together, our results provide insights into how the high-affinity end-binding of EB1 is achieved and how this activity may be regulated in cells.

Short Direct Repeats in the 3' Untranslated Region Are Involved in Subgenomic Flaviviral RNA Production.

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation.IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5'-3' exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3'-UTR. The 3'-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.