Identification of Rhopalosiphum Padi Virus 5′ Untranslated Region Sequences Required for Cryptic Promoter Activity and Internal Ribosome Entry

The 579-nucleotide 5′ untranslated region (5′UTR) of the Rhopalosiphum padi virus (RhPV) possesses a cross-kingdom internal ribosome entry site (IRES) activity that functions in insect, mammalian, and plant-derived in vitro translation systems, and six TAAG motifs within the DNA fragment encoding the RhPV 5′UTR were previously found to confer the RhPV 5′UTR with late promoter activity in baculovirus. In the present study, various truncated RhPV 5′UTR sequences were produced, and among them, a fragment of 110 bp ranging from nucleotides 309 to 418 was identified to be the shortest fragment responsible for the late promoter activity in baculovirus infected Sf21 cells. This 110 bp fragment contains a TAAG tandem repeat that retains more than 60% of the late promoter activity of the full length RhPV 5′UTR sequence. Further, IRES activity remained unchanged in all truncated RhPV 5′UTR constructs. Taken together, this novel 110 bp fragment having late promoter activity in baculovirus as well as IRES activity in mammalian cell, renders it a useful tool for the development of a “shuttle” bi-cistronic baculovirus gene expression and/or delivery vector.     

Circular RNA,the Key for Translation

It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m⁶A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5′ end-independent translation initiation. Today a new family of so-called “noncoding” circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5′ end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3′–5′ interaction have been reported, including m⁶A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and noncovalently closed circular mRNA translation landscape shows that RNA with circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.     

Interruptions of the FXN GAA Repeat Tract Delay the Age at Onset of Friedreich’s Ataxia in a Location Dependent Manner

Friedreich’s ataxia (FRDA) is a comparatively rare autosomal recessive neurological disorder primarily caused by the homozygous expansion of a GAA trinucleotide repeat in intron 1 of the FXN gene. The repeat expansion causes gene silencing that results in deficiency of the frataxin protein leading to mitochondrial dysfunction, oxidative stress and cell death. The GAA repeat tract in some cases may be impure with sequence variations called interruptions. It has previously been observed that large interruptions of the GAA repeat tract, determined by abnormal MboII digestion, are very rare. Here we have used triplet repeat primed PCR (TP PCR) assays to identify small interruptions at the 5′ and 3′ ends of the GAA repeat tract through alterations in the electropherogram trace signal. We found that contrary to large interruptions, small interruptions are more common, with 3′ interruptions being most frequent. Based on detection of interruptions by TP PCR assay, the patient cohort (n = 101) was stratified into four groups: 5′ interruption, 3′ interruption, both 5′ and 3′ interruptions or lacking interruption. Those patients with 3′ interruptions were associated with shorter GAA1 repeat tracts and later ages at disease onset. The age at disease onset was modelled by a group-specific exponential decay model. Based on this modelling, a 3′ interruption is predicted to delay disease onset by approximately 9 years relative to those lacking 5′ and 3′ interruptions. This highlights the key role of interruptions at the 3′ end of the GAA repeat tract in modulating the disease phenotype and its impact on prognosis for the patient.     

Molecular Dissection of Escherichia coli CpdB: Roles of the N Domain in Catalysis and Phosphate Inhibition,and of the C Domain in Substrate Specificity and Adenosine Inhibition

CpdB is a 3′-nucleotidase/2′3′-cyclic nucleotide phosphodiesterase, active also with reasonable efficiency on cyclic dinucleotides like c-di-AMP (3′,5′-cyclic diadenosine monophosphate) and c-di-GMP (3′,5′-cyclic diadenosine monophosphate). These are regulators of bacterial physiology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-β response in infected hosts. The cpdB gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors. Their protein products are extracytoplasmic enzymes (either periplasmic or cell–wall anchored) and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3′-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2′,3′-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site.     

Human DDX3X Unwinds Japanese Encephalitis and Zika Viral 5′ Terminal Regions

Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5′ terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5′ TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5′ TR of JEV and demonstrated its direct interaction with recombinant DDX3X (K_d of 1.66 ± 0.21 µM) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5′ and 3′ TRs of flaviviruses, we investigated if the ZIKV 5′ TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5′ TR with a K_d of 7.05 ± 0.75 µM. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5′ TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication     

Phenylalanine-Derived β-Lactam TRPM8 Modulators. Configuration Effect on the Antagonist Activity

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca²⁺ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic β–lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3′-phenyl-2′-dibenzylamino)prop-1′-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca²⁺ entry to larger or lesser extent. Potency follows the order 3R,4R,2′R > 3S,4S,2′R ≅ 3R,4R,2′S > 3S,4S,2′S, with the most potent diastereoisomer showing a half inhibitory concentration (IC₅₀) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these β-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.     

Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.