Identification of RNA structures and their roles in RNA functions.

The development of high-throughput RNA structure profiling methods in the past decade has greatly facilitated our ability to map and characterize different aspects of RNA structures transcriptome-wide in cell populations, single cells and single molecules. The resulting high-resolution data have provided insights into the static and dynamic nature of RNA structures, revealing their complexity as they perform their respective functions in the cell. In this Review, we discuss recent technical advances in the determination of RNA structures, and the roles of RNA structures in RNA biogenesis and functions, including in transcription, processing, translation, degradation, localization and RNA structure-dependent condensates. We also discuss the current understanding of how RNA structures could guide drug design for treating genetic diseases and battling pathogenic viruses, and highlight existing challenges and future directions in RNA structure research.

Core-binding factor fusion downregulation of ADAR2 RNA editing contributes to AML leukemogenesis.

Adenosine-to-inosine RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes, ADAR1 and ADAR2, has been shown to contribute to multiple cancers. However, other than the chronic myeloid leukemia blast crisis, relatively little is known about its role in other types of hematological malignancies. Here, we found that ADAR2, but not ADAR1 and ADAR3, was specifically downregulated in the core-binding factor (CBF) acute myeloid leukemia (AML) with t(8;21) or inv(16) translocations. In t(8;21) AML, RUNX1-driven transcription of ADAR2 was repressed by the RUNX1-ETO additional exon 9a fusion protein in a dominant-negative manner. Further functional studies confirmed that ADAR2 could suppress leukemogenesis specifically in t(8;21) and inv16 AML cells dependent on its RNA editing capability. Expression of 2 exemplary ADAR2-regulated RNA editing targets coatomer subunit α and component of oligomeric Golgi complex 3 inhibits the clonogenic growth of human t(8;21) AML cells. Our findings support a hitherto, unappreciated mechanism leading to ADAR2 dysregulation in CBF AML and highlight the functional relevance of loss of ADAR2-mediated RNA editing to CBF AML.

Non-coding RNA LEVER sequestration of PRC2 can mediate long range gene regulation.

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator required for gene silencing during development. Although PRC2 is a well-established RNA-binding complex, the biological function of PRC2-RNA interaction has been controversial. Here, we study the gene-regulatory role of the inhibitory PRC2-RNA interactions. We report a nuclear long non-coding RNA, LEVER, which mapped 236 kb upstream of the ß-globin cluster as confirmed by Nanopore sequencing. LEVER RNA interacts with PRC2 in its nascent form, and this prevents the accumulation of the H3K27 repressive histone marks within LEVER locus. Interestingly, the accessible LEVER chromatin, in turn, suppresses the chromatin interactions between the ε-globin locus and ß-globin locus control region (LCR), resulting in a repressive effect on ε-globin gene expression. Our findings validate that the nascent RNA-PRC2 interaction inhibits local PRC2 function in situ. More importantly, we demonstrate that such a local process can in turn regulate the expression of neighboring genes.

Evaluation of Food Safety and Quality Parameters for Shelf Life Extension of Pulsed Light Treated Strawberries.

Strawberry is a healthy fruit with numerous health-benefit compounds. Unfortunately, it is highly perishable and occasionally can be contaminated with foodborne pathogens. The overall goal of this study is to evaluate pulsed light (PL) processing for disinfection of strawberries, extension of shelf life, and preservation of quality attributes and compounds that are beneficial to health. Preliminary screening of PL conditions based on visual appearance of strawberries was conducted, and 3 PL treatments were identified for full evaluation. Salmonella inoculum was artificially deposited onto the skin of strawberries via spot-inoculation or dip-inoculation. The 3 PL treatments slightly reduced the level of inoculated Salmonella on strawberries, ranging from approximately 0.4 to 0.8 log reduction. They also slowed down the visible mold development on strawberries by 2 to 4 days compared with the untreated control. Regarding the natural yeasts and molds, the quality attributes (weight loss and firmness), and the bioactive compounds (total anthocyanin, total phenolics, and total antioxidant activity). The 3 PL treatment showed no significant or negligible difference comparing to the control group. Overall, the 3 PL treatments demonstrated potential in extending the shelf life of strawberries. The quality attributes or the bioactive compounds of strawberries showed no significant or minimal change after these PL treatments. PRACTICAL APPLICATION: Pulsed light (PL) processing for strawberry decontamination and shelf life extension was evaluated. Results demonstrated that PL processing could have the potency to improve strawberry shelf life without significantly affecting the quality and bioactive compounds of strawberries.

Evaluation of pulsed light treatments on inactivation of Salmonella on blueberries and its impact on shelf-life and quality attributes.

Blueberry have a short shelf life when fully ripe and susceptible to contamination of various pathogens. Our study investigated the effect of pulsed light (PL) on inactivation of Salmonella on blueberries and its impact on shelf-life, quality attributes and health-benefit compounds of blueberries. Dry PL (6J/cm2) and water-assisted PL (samples were agitated in water during PL treatment; 9J/cm2) along with two controls, dry control (untreated) and water-assisted control (water washing without PL), were applied to blueberries with subsequent storages at room temperature (3days) or 5°C (7days). For Salmonella inactivation, dry PL treatment achieved 0.9 and 0.6 log reduction of Salmonella for spot and dip inoculation, respectively; while the water-assisted PL treatment reduced Salmonella by 4.4 log and 0.8 log for spot and dip inoculation, respectively. The water-assisted PL treatment resulted in Salmonella populations significantly lower than the dry control after storage regardless of the storage temperature and inoculation method. Neither dry nor water-assisted PL treatments improved the shelf life of blueberries even though direct inactivation of natural yeasts and molds were achieved. Surface lightness was instantly reduced after both dry and water-assisted PL treatments. Compared with the dry control, the two PL treatments did not reduce the firmness of blueberries. Weight loss was increased for the dry PL treated samples, but not for the water-assisted PL treatment for both storage conditions. Delayed anthocyanins accumulation and reduced total antioxidant activity were induced by both PL treatments at the end of storage at room temperature, while slight enhancement in total phenolics content was achieved by water-assisted PL treatment. In conclusion, the water-assisted PL treatment could effectively decontaminate Salmonella on blueberries while showed minimal or no impact on the shelf-life, quality attributes and health-benefit compounds of blueberries. PL processing parameters need to be further evaluated and optimized before possible application in the blueberry industry.

Pulsed light inactivation of murine norovirus, Tulane virus, Escherichia coli O157:H7 and Salmonella in suspension and on berry surfaces.

Pulsed light (PL) inactivation of two human norovirus (HuNoV) surrogates, murine norovirus (MNV-1) and Tulane virus (TV), and two bacterial pathogens, Escherichia coli O157:H7 and Salmonella, were evaluated. The viruses and bacteria were suspended in phosphate buffered saline (PBS) to final populations of ∼6 log PFU/mL and ∼6 log CFU/mL, respectively. Both viral and bacterial suspensions were then irradiated by PL for different durations and the reductions of each microorganisms were determined. MNV-1 and TV were significantly (P < 0.05) more resistant to PL treatment than Salmonella and E. coli O157:H7 in PBS suspension. MNV-1, Salmonella and E. coli O157:H7 were also inoculated on strawberries and blueberries and the PL inactivation of each microorganism was determined. Lower inactivation of each microorganism was achieved on berry surfaces than in PBS suspension. This study shows that PL can induce rapid inactivation of MNV-1, TV, Salmonella and E. coli O157:H7 in clear suspension with viruses more resistant to PL treatment than bacteria. The efficacy of PL treatment is substantially influenced by food surface structure.

Quantitatively relating gene expression to light intensity via the serial connection of blue light sensor and CRISPRi.

The ability to regulate endogenous gene expression is critical in biological research. Existing technologies, such as RNA interference, zinc-finger regulators, transcription-activator-like effectors, and CRISPR-mediated regulation, though proved to be competent in significantly altering expression levels, do not provide a quantitative adjustment of regulation effect. As a solution to this problem, we place CRISPR-mediated interference under the control of blue light: while dCas9 protein is constitutively expressed, guide RNA transcription is regulated by YF1-FixJ-PFixK2, a blue light responding system. With a computer-controlled luminous device, the quantitative relationship between target gene expression and light intensity has been determined. As the light intensifies, the expression level of target gene gradually ascends. This remarkable property enables sensor-CRISPRi to accurately interrogate cellular activities.