Nuclear Magnetic Resonance reveals a two hairpin equilibrium near the 3'-splice site of Influenza A segment 7 mRNA that can be shifted by oligonucleotides

Influenza A kills hundreds of thousands of people globally every year and has potential to generate more severe pandemics. Influenza A’s RNA genome and transcriptome provide many potential therapeutic targets. Here, nuclear magnetic resonance (NMR) experiments suggest that one such target could be a hairpin loop of eight nucleotides in a pseudoknot that sequesters a 3' splice site in canonical pairs until a conformational change releases it into a dynamic 2X2 nucleotide internal loop. NMR experiments reveal that the hairpin loop is dynamic and able to bind oligonucleotides as short as pentamers. A 3D NMR structure of the complex contains four and likely five base pairs between pentamer and loop. Moreover, a hairpin sequence was discovered that mimics the equilibrium of the influenza hairpin between its structure in the pseudoknot and upon release of the splice site. Oligonucleotide binding shifts the equilibrium completely to the hairpin secondary structure required for pseudoknot folding. The results suggest this hairpin can be used to screen for compounds that stabilize the pseudoknot and potentially reduce splicing.

Error feedback regulation for 1D anti-stable wave equation

This paper is concerned with the boundary error feedback regulation for a one-dimensional anti-stable wave equation with distributed disturbance generated by a finite-dimensional exogenous system. Transport equation and regulator equation are introduced first to deal with the anti-damping on boundary and the distributed disturbance of the original system. Then, the tracking error and its derivative are measured to design an observer for both exosystem and auxiliary partial differential equation (PDE) system to recover the state. After proving the well-posedness of the regulator equations, we propose an observer-based controller to regulate the tracking error to zero exponentially and keep the states of all the internal loop uniformly bounded. Finally, some numerical simulations are presented to validate the effectiveness of the proposed controller.

The conserved single-cleavage mechanism of animal DROSHA enzymes

RNase III enzymes typically cleave both strands of double-stranded RNAs (dsRNAs). We recently discovered that a human RNase III, DROSHA, exhibits a single cleavage on the one strand of primary microRNAs (pri-miRNAs). This study revealed that DROSHAs from the other animals, including worms and flies, also show the single cleavage on dsRNAs. Furthermore, we demonstrated that the mechanism of single cleavage is conserved in animal DROSHA enzymes. In addition, the dsRNA-binding domain (dsRBD) and a 3p-strand cleavage-supporting helix (3pCSH) of the DROSHA enzymes foster a weak single cleavage on one strand, which ensures their double cleavages. Disrupting the interaction of dsRBD-RNA and 3pCSH-RNA by an internal loop (IL) and a 3pCSH-loop in the lower stem of pri-miRNAs, respectively, inhibits one of the double cleavages of DROSHAs, and this results in the single cleavage. Our findings expand our understanding of the enzymatic mechanisms of animal DROSHAs. They also indicate that there are currently unknown cellular functions of DROSHA enzymes using their single cleavage activity.

1H, 13C and 15N assignment of stem-loop SL1 from the 5'-UTR of SARS-CoV-2

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.

The neural G protein Gαo tagged with GFP at an internal loop is functional in C. elegans

Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to complement the already well-developed genetic analysis of Gαo signaling.