Background/Aims: RNA elements such as catalytic RNA, riboswitch, microRNA, and long non coding RNA (lncRNA) play central roles in many cellular processes. Studying diverse RNA functions require large quantities of RNA for precise structure analysis. Current RNA structure and function studies can benefit from improved RNA quantity and quality, simpler separation procedure and enhanced accuracy of structural analysis. Methods: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion chromatography (SEC) based denaturing purification and improved secondary structure analysis by chemical probing. Results: We observed that higher Mg²⁺, nucleoside triphosphate (NTP) concentrations and longer reaction duration can improve the RNA yield from in vitro transcription, specifically for longer and more complicated constructs. Our improved SEC-based denaturing RNA purification effectively halved the experiment duration and labor without introducing any contaminant. Finally, this study increased the accuracy and signal-to-noise ratio (SNR) of selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing for analyzing RNA structure. Conclusion: Part or all of our modified method can improve almost any RNA-related study from protein-RNA interaction analysis to crystallography.

Phosphate, a key plant nutrient, is perceived through inositol polyphosphates (InsPs) by SPX domain-containing proteins. SPX1 an inhibit the PHR2 transcription factor to maintain Pi homeostasis. How SPX1 recognizes an InsP molecule and represses transcription activation by PHR2 remains unclear. Here we show that, upon binding InsP6, SPX1 can disrupt PHR2 dimers and form a 1:1 SPX1-PHR2 complex. The complex structure reveals that SPX1 helix α1 can impose a steric hindrance when interacting with the PHR2 dimer. By stabilizing helix α1, InsP6 allosterically decouples the PHR2 dimer and stabilizes the SPX1-PHR2 interaction. In doing so, InsP6 further allows SPX1 to engage with the PHR2 MYB domain and sterically block its interaction with DNA. Taken together, our results suggest that, upon sensing the surrogate signals of phosphate, SPX1 inhibits PHR2 via a dual mechanism that attenuates dimerization and DNA binding activities of PHR2.

Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA–gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a ‘Z’-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.

Myocardial infarction is irreversible cardiac tissue necrosis due to the blockage of one of the arteries. It leads to an insufficient supply of oxygen and nutrients, creating muscular damage in the affected regions. In the present study, aqueous methanolic extract of Thymus linearis was prepared to evaluate its activity against ischemic stress due to free radical production. GC−MS analysis was performed to evaluate the phytochemicals present in the plant extract. A chemical database of 30 compounds was virtually screened against NF-κB, COX2, and MCL, where γ-cadinene, β-bisabolene, and β-caryophyllene were found to be the best interacting ligands. To systematically assess cardioprotective activity against ischemia, isoproterenol and doxorubicin were used to induce cardiotoxicity in rats. The prepared extract of T. linearis (100 mg/kg) was given daily to animals for 21 days before injecting isoproterenol (85 mg/kg of animal weight) subcutaneously in two doses on the 20th and 21st days. In the case of doxorubicin, cardiotoxicity was induced in rats by a single injection (15 mg/ kg) on the seventh day, and the extract was given to animals for 10 consecutive days. Animals' blood samples were used to monitor cardiac, liver, and other marker enzymes, including LDH, CPK, and AST. Superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were also assayed in blood plasma to determine the degree of oxidative stress. H&E staining was performed to evaluate cardioprotection by plant extract, showing significant preventive effects in reducing cardiac injury induced by isoproterenol and doxorubicin.

Riboswitches are regulatory noncoding RNAs found in bacteria, fungi and plants, that modulate gene expressions through structural changes in response to ligand binding. Understanding how ligands interact with riboswitches in solution can shed light on the molecular mechanisms of this ancient regulators. Previous studies showed that riboswitches undergo global conformation changes in response to ligand binding to relay information. Here, we report conformation switching models of the recently discovered tetrahydrofolic acid-responsive second class of tetrahydrofolate (THF-II) riboswitches in response to ligand binding. Using a combination of selective 2′-hydroxyl acylation, analyzed by primer extension (SHAPE) assay, 3D modeling and small-angle X-ray scattering (SAXS), we found that the ligand specifically recognizes and reshapes the THF-II riboswitch loop regions, but does not affect the stability of the P3 helix. Our results show that the THF-II riboswitch undergoes only local conformation changes in response to ligand binding, rearranging the Loop1-P3-Loop2 region and rotating Loop1 from a ~120° angle to a ~75° angle. This distinct conformation changes suggest a unique regulatory mechanism of the THF-II riboswitch, previously unseen in other riboswitches. Our findings may contribute to the fields of RNA sensors and drug design.