Distinct structural bases for sequence-specific DNA binding by mammalian BEN domain proteins.

The BEN domain is a recently recognized DNA binding module that is present in diverse metazoans and certain viruses. Several BEN domain factors are known as transcriptional repressors, but, overall, relatively little is known of how BEN factors identify their targets in humans. In particular, X-ray structures of BEN domain:DNA complexes are only known for Drosophila factors bearing a single BEN domain, which lack direct vertebrate orthologs. Here, we characterize several mammalian BEN domain (BD) factors, including from two NACC family BTB-BEN proteins and from BEND3, which has four BDs. In vitro selection data revealed sequence-specific binding activities of isolated BEN domains from all of these factors. We conducted detailed functional, genomic, and structural studies of BEND3. We show that BD4 is a major determinant for in vivo association and repression of endogenous BEND3 targets. We obtained a high-resolution structure of BEND3-BD4 bound to its preferred binding site, which reveals how BEND3 identifies cognate DNA targets and shows differences with one of its non-DNA-binding BEN domains (BD1). Finally, comparison with our previous invertebrate BEN structures, along with additional structural predictions using AlphaFold2 and RoseTTAFold, reveal distinct strategies for target DNA recognition by different types of BEN domain proteins. Together, these studies expand the DNA recognition activities of BEN factors and provide structural insights into sequence-specific DNA binding by mammalian BEN proteins.

Structure-based characterization and compound identification of the wild-type THF class-II riboswitch.

Riboswitches are conserved regulatory RNA elements participating in various metabolic pathways. Recently, a novel RNA motif known as the folE RNA motif was discovered upstream of folE genes. It specifically senses tetrahydrofolate (THF) and is therefore termed THF-II riboswitch. To unravel the ligand recognition mechanism of this newly discovered riboswitch and decipher the underlying principles governing its tertiary folding, we determined both the free-form and bound-form THF-II riboswitch in the wild-type sequences. Combining structural information and isothermal titration calorimetry (ITC) binding assays on structure-based mutants, we successfully elucidated the significant long-range interactions governing the function of THF-II riboswitch and identified additional compounds, including alternative natural metabolites and potential lead compounds for drug discovery, that interact with THF-II riboswitch. Our structural research on the ligand recognition mechanism of the THF-II riboswitch not only paves the way for identification of compounds targeting riboswitches, but also facilitates the exploration of THF analogs in diverse biological contexts or for therapeutic applications.

Hatchet ribozyme structure and implications for cleavage mechanism.

Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5' nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.

Increase in economic efficiency of water use caused by crop structure adjustment in arid areas.

The Heihe River is located in the arid zone of northwestern China. In its middle-reach region, irrigation agriculture is well developed. With rapid population growth and expansion of the cultivated land in this region, effective water resource use is vital for the sustainable development of the river basin and the increase of incomes from farming practices. In this study, based on farmer survey data, the input parameters of the CROPWAT model were modified, the water use amount was simulated after deducting the influences of climate, seeds, and irrigation systems, and the variation of economic efficiency of water use (EEWU) induced by crop structure adjustment from 2001 to 2012 was analyzed. The results show that simulations for evapotranspiration of maize based on the CROPWAT model are in accord with the observed data. From 2001 to 2012, due to changes in the regional crop structure, EEWU in the study area increased by about 40%. In the arid areas in northwest China, crop structure adjustment has a huge potential for improving EEWU and increasing incomes from farming practices.

Structure-based insights into recognition and regulation of SAM-sensing riboswitches.

Riboswitches are highly conserved RNA elements that located in the 5'-UTR of mRNAs, which undergo real-time structure conformational change to achieve the regulation of downstream gene expression by sensing their cognate ligands. S-adenosylmethionine (SAM) is a ubiquitous methyl donor for transmethylation reactions in all living organisms. SAM riboswitch is one of the most abundant riboswitches that bind to SAM with high affinity and selectivity, serving as regulatory modules in multiple metabolic pathways. To date, seven SAM-specific riboswitch classes that belong to four families, one SAM/SAH riboswitch and one SAH riboswitch have been identified. Each SAM riboswitch family has a well-organized tertiary core scaffold to support their unique ligand-specific binding pocket. In this review, we summarize the current research progress on the distribution, structure, ligand recognition and gene regulation mechanism of these SAM-related riboswitch families, and further discuss their evolutionary prospects and potential applications.

High-sensitivity profiling of SARS-CoV-2 noncoding region-host protein interactome reveals the potential regulatory role of negative-sense viral RNA.

A deep understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-host interactions is crucial to developing effective therapeutics and addressing the threat of emerging coronaviruses. The role of noncoding regions of viral RNA (ncrRNAs) has yet to be systematically scrutinized. We developed a method using MS2 affinity purification coupled with liquid chromatography-mass spectrometry and designed a diverse set of bait ncrRNAs to systematically map the interactome of SARS-CoV-2 ncrRNA in Calu-3, Huh7, and HEK293T cells. Integration of the results defined the core ncrRNA-host protein interactomes among cell lines. The 5' UTR interactome is enriched with proteins in the small nuclear ribonucleoproteins family and is a target for the regulation of viral replication and transcription. The 3' UTR interactome is enriched with proteins involved in the stress granules and heterogeneous nuclear ribonucleoproteins family. Intriguingly, compared with the positive-sense ncrRNAs, the negative-sense ncrRNAs, especially the negative-sense of 3' UTR, interacted with a large array of host proteins across all cell lines. These proteins are involved in the regulation of the viral production process, host cell apoptosis, and immune response. Taken together, our study depicts the comprehensive landscape of the SARS-CoV-2 ncrRNA-host protein interactome and unveils the potential regulatory role of the negative-sense ncrRNAs, providing a new perspective on virus-host interactions and the design of future therapeutics. Given the highly conserved nature of UTRs in positive-strand viruses, the regulatory role of negative-sense ncrRNAs should not be exclusive to SARS-CoV-2. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic affecting millions of lives. During replication and transcription, noncoding regions of the viral RNA (ncrRNAs) may play an important role in the virus-host interactions. Understanding which and how these ncrRNAs interact with host proteins is crucial for understanding the mechanism of SARS-CoV-2 pathogenesis. We developed the MS2 affinity purification coupled with liquid chromatography-mass spectrometry method and designed a diverse set of ncrRNAs to identify the SARS-CoV-2 ncrRNA interactome comprehensively in different cell lines and found that the 5' UTR binds to proteins involved in U1 small nuclear ribonucleoprotein, while the 3' UTR interacts with proteins involved in stress granules and the heterogeneous nuclear ribonucleoprotein family. Interestingly, negative-sense ncrRNAs showed interactions with a large number of diverse host proteins, indicating a crucial role in infection. The results demonstrate that ncrRNAs could serve diverse regulatory functions.

Strategies for understanding RNA recognition by X-ray crystallography and NMR methods.

One class of non-coding RNA molecules, termed riboswitches, are the regulatory elements with ability to control gene expression by sensing different cellular metabolites. Specific recognition of the corresponding ligand induced the conformation rearrangement of riboswitch, and thus turned on/off the down-stream gene expression. In this chapter, we will focus on two principal methods that are currently used to investigate the recognition mechanism of riboswitches, including X-ray crystallography and NMR spectroscopy. First, we present that how to get the diffraction-quality crystals of riboswitches in specific functional states. Then we focus on how to use NMR spectroscopy as a tool to learn the dynamics of riboswitches. Detailed protocols are listed below.

Structure-based insights into self-cleavage by a four-way junctional twister-sister ribozyme.

Here we report on the crystal structure and cleavage assays of a four-way junctional twister-sister self-cleaving ribozyme. Notably, 11 conserved spatially separated loop nucleotides are brought into close proximity at the ribozyme core through long-range interactions mediated by hydrated Mg2+ cations. The C62-A63 step at the cleavage site adopts a splayed-apart orientation, with flexible C62 directed outwards, whereas A63 is directed inwards and anchored by stacking and hydrogen-bonding interactions. Structure-guided studies of key base, sugar, and phosphate mutations in the twister-sister ribozyme, suggest contributions to the cleavage chemistry from interactions between a guanine at the active site and the non-bridging oxygen of the scissile phosphate, a feature found previously also for the related twister ribozyme. Our four-way junctional pre-catalytic structure differs significantly in the alignment at the cleavage step (splayed-apart vs. base-stacked) and surrounding residues and hydrated Mg2+ ions relative to a reported three-way junctional pre-catalytic structure of the twister-sister ribozyme.