RNase H-based analysis of synthetic mRNA 5' cap incorporation.

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass analysis. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.

Molecular changes in Mesembryanthemum crystallinum guard cells underlying the C3 to CAM transition.

ABSTARCT: KEY MESSAGE: The timing and transcriptomic changes during the C3 to CAM transition of common ice plant support the notion that guard cells themselves can shift from C3 to CAM. Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis: stomata close during the day, enhancing water conservation, and open at night, allowing CO2 uptake. Mesembryanthemum crystallinum (common ice plant) is a facultative CAM species that can shift from C3 photosynthesis to CAM under salt or drought stresses. However, the molecular mechanisms underlying the stress induced transition from C3 to CAM remain unknown. Here we determined the transition time from C3 to CAM in M. crystallinum under salt stress. In parallel, single-cell-type transcriptomic profiling by 3'-mRNA sequencing was conducted in isolated stomatal guard cells to determine the molecular changes in this key cell type during the transition. In total, 495 transcripts showed differential expression between control and salt-treated samples during the transition, including 285 known guard cell genes, seven CAM-related genes, 18 transcription factors, and 185 other genes previously not found to be expressed in guard cells. PEPC1 and PPCK1, which encode key enzymes of CAM photosynthesis, were up-regulated in guard cells after seven days of salt treatment, indicating that guard cells themselves can shift from C3 to CAM. This study provides important information towards introducing CAM stomatal behavior into C3 crops to enhance water use efficiency.

Environmentally-triggered contraction of the norovirus virion determines diarrheagenic potential.

Noroviruses are the leading cause of severe childhood diarrhea and foodborne disease worldwide. While they are a major cause of disease in all age groups, infections in the very young can be quite severe with annual estimates of 50,000-200,000 fatalities in children under 5 years old. In spite of the remarkable disease burden associated with norovirus infections in people, very little is known about the pathogenic mechanisms underlying norovirus diarrhea, principally because of the lack of tractable small animal models. We recently demonstrated that wild-type neonatal mice are susceptible to murine norovirus (MNV)-induced acute self-resolving diarrhea in a time course mirroring human norovirus disease. Using this robust pathogenesis model system, we demonstrate that virulence is regulated by the responsiveness of the viral capsid to environmental cues that trigger contraction of the VP1 protruding (P) domain onto the particle shell, thus enhancing receptor binding and infectivity. The capacity of a given MNV strain to undergo this contraction positively correlates with infection of cells expressing low abundance of the virus receptor CD300lf, supporting a model whereby virion contraction triggers infection of CD300lflo cell types that are responsible for diarrhea induction. These findings directly link environmentally-influenced biophysical features with norovirus disease severity.

Physiological Changes in Mesembryanthemum crystallinum During the C3 to CAM Transition Induced by Salt Stress.

Salt stress impedes plant growth and development, and leads to yield loss. Recently, a halophyte species Mesembryanthemum crystallinum has become a model to study plant photosynthetic responses to salt stress. It has an adaptive mechanism of shifting from C3 photosynthesis to crassulacean acid metabolism (CAM) photosynthesis under stresses, which greatly enhances water usage efficiency and stress tolerance. In this study, we focused on investigating the morphological and physiological changes [e.g., leaf area, stomatal movement behavior, gas exchange, leaf succulence, and relative water content (RWC)] of M. crystallinum during the C3 to CAM photosynthetic transition under salt stress. Our results showed that in M. crystallinum seedlings, CAM photosynthesis was initiated after 6 days of salt treatment, the transition takes place within a 3-day period, and plants became mostly CAM in 2 weeks. This result defined the transition period of a facultative CAM plant, laid a foundation for future studies on identifying the molecular switches responsible for the transition from C3 to CAM, and contributed to the ultimate goal of engineering CAM characteristics into C3 crops.