An origin of the immunogenicity of in vitro transcribed RNA

Abstract The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg2+ concentration. The current work thus not only offers a previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system, but also shows that the immune-stimulatory activity can be readily regulated

Rare earths complexes of single arm N-aryl Schiff base ligand

A synthesized ligand N – ( 2 – hydroxyphenyl) – 4 – n – butylsalicylaldimine (H2L) was used to prepare a series of mononuclear lanthanide complexes of the type [LnH2L(HL)2Cl] (Ln =LaIII, CeIII, SmIII and GdIII). The ligand and complexes were characterized by 1H, 13CNMR, elemental analysis (CHNO), FT-IR spectroscopy, GC-mass, Magnetic susceptibility Molar Conductivity and Thermo gravimetric analysis (TGA). Investigation of complexes suggest that two ligand molecules behaves as tridentate and the third one as bidentate as it present in zwitter ionic form with an additional one chloride ion to complete nine coordination geometry

Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity.

The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self versus non-self nucleic acid discrimination

Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

<div><p>D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD’s invariant, cross-subunit Gly-<i>cis</i>Pro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNA^Gly. High-resolution crystal structure reveals that the architecture of DTD’s chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNA^Gly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNA^Gly. Our in vitro observations are substantiated by cell-based studies in <i>Escherichia coli</i> that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2′-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD’s activity on Gly-tRNA^Gly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting.</p></div

ADAR1 prevents autoinflammation by suppressing spontaneous ZBP1 activation

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)(.) In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutieres syndrome (AGS). In mice, complete loss of ADAR1 activity is embryonically lethal, and mutations similar to those found in patients with AGS cause autoinflammation. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Z alpha domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Z alpha domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function

Model for protection of Gly-tRNA^Gly by EF-Tu.

<p>In the cell, the aminoacyl-tRNA pool comprises mostly L-aminoacyl-tRNAs, some Gly-tRNA^Gly, and very few D-aminoacyl-tRNAs (central circle). L-aminoacyl-tRNAs are not acted upon by DTD and, hence, no editing (top left). DTD efficiently decouples D-amino acids mischarged on tRNAs (chiral proofreading), even in the presence of abundant EF-Tu (top right). Gly-tRNA^Gly can be edited by DTD in the absence of EF-Tu (misediting; bottom left), which is, however, effectively prevented by EF-Tu (protection from misediting; bottom right). Under conditions in which DTD levels are relatively high, i.e., DTD is overexpressed, protection is relieved, leading to glycine misediting and cellular toxicity (bottom middle).</p

Glycine misediting by DTD is a universal phenomenon.

<p><b>(a)</b> Structure-based multiple sequence alignment of DTD from various organisms highlighting the variant residues in the active site. Residues marked with stars are within 6 Å radius of D-Tyr moiety of D-Tyr3AA. Amino acids enclosed in green boxes are varying in DTDs across different organisms. DTDs from organisms indicated by black arrowheads have been tested for biochemical activity in the current study. <b>(b)</b> Non-conserved residues in the chiral proofreading site of various DTDs—<i>Plasmodium falciparum</i> (green; PDB id: 4NBI), <i>Escherichia coli</i> (violet; PDB id: 1JKE), <i>Leishmania major</i> (cyan; model), <i>Drosophila melanogaster</i> (yellow; model), and <i>Danio rerio</i> (pink; model). <b>(c)</b> Deacylation of Gly-tRNA^Gly by buffer (blue circle), 50 nM LmDTD (green circle), 50 nM DmDTD (red square), and 50 nM DrDTD (brown triangle). Error bars indicate one standard deviation from the mean. The underlying data of panel <b>(c)</b> can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002465#pbio.1002465.s001" target="_blank">S1 Data</a>.</p

Crystallographic data collection and refinement statistics.

<p>Crystallographic data collection and refinement statistics.</p