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1. DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification

   https://doi.org/10.1038/s41467-020-16321-6  

   Lentini, Jenna M. ; Alsaif, Hessa S. ; Faqeih, Eissa ; Alkuraya, Fowzan S. ; Fu, Dragony ( May 2020 , Nature Communications)

   In mammals, a subset of arginine tRNA isoacceptors are methylated in the anticodon loop by the METTL2 methyltransferase to form the 3-methylcytosine (m3C) modification. However, the mechanism by which METTL2 identifies specific tRNA arginine species for m3C formation as well as the biological role of m3C in mammals is unknown. Here, we show that human METTL2 forms a complex with DALR anticodon binding domain containing 3 (DALRD3) protein to recognize particular arginine tRNAs destined for m3C modification. DALRD3-deficient human cells exhibit nearly complete loss of the m3C modification in tRNA-Arg species. Notably, we identify a homozygous nonsense mutation in theDALRD3gene that impairs m3C formation in human patients exhibiting developmental delay and early-onset epileptic encephalopathy. These findings uncover an unexpected function for the DALRD3 protein in the targeting of distinct arginine tRNAs for m3C modification and suggest a crucial biological role for DALRD3-dependent tRNA modification in proper neurological development.

    

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2. Experimental and theoretical rationalization for the base pairing abilities of inosine, guanosine, adenosine, and their corresponding 8‐oxo ‐7,8‐dihydropurine, and 8‐bromopurine analogues within A‐form duplexes of RNA

   https://doi.org/10.1002/bip.23410  

   Skinner, Austin ; Yang, Chou‐Hsun ; Hincks, Kazuki ; Wang, Haobin ; Resendiz, Marino J. E. ( November 2020 , Biopolymers)

   Inosine is an important RNA modification, furthermore RNA oxidation has gained interest due, in part, to its potential role in the development/progression of disease as well as on its impact on RNA structure and function. In this report we established the base pairing abilities of purine nucleobases G, I, A, as well as their corresponding, 8‐oxo‐7,8‐dihydropurine (common products of oxidation at the C8‐position of purines), and 8‐bromopurine (as probes to explore conformational changes), derivatives, namely 8‐oxoG, 8‐oxoI, 8‐oxoA, 8‐BrG, and 8‐BrI. Dodecamers of RNA were obtained using standard phosphoramidite chemistry via solid‐phase synthesis, and used as models to establish the impact that each of these nucleobases have on the thermal stability of duplexes, when base pairing to canonical and noncanonical nucleobases. Thermal stabilities were obtained from thermal denaturation transition (T_m) measurements, via circular dichroism (CD). The results were then rationalized using models of base pairs between two monomers, via density functional theory (DFT), that allowed us to better understand potential contributions from H‐bonding patterns arising from distinct conformations. Overall, some of the important results indicate that: (a) an anti‐I:syn‐A base pair provides thermal stability, due to the absence of the exocyclic amine; (b) 8‐oxoG base pairs like U, and does not induce destabilization within the duplex when compared to the pyrimidine ring; (c) a U:G wobble‐pair is only stabilized by G; and (d) 8‐oxoA displays an inherited base pairing promiscuity in this sequence context. Gaining a better understanding of how this oxidatively generated lesions potentially base pair with other nucleobases will be useful to predict various biological outcomes, as well as in the design of biomaterials and/or nucleotide derivatives with biological potential.

    

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3. A mouse model of seizures in anti– N ‐methyl‐ d ‐aspartate receptor encephalitis

   https://doi.org/10.1111/epi.14662  

   Taraschenko, Olga ; Fox, Howard S. ; Pittock, Sean J. ; Zekeridou, Anastasia ; Gafurova, Maftuna ; Eldridge, Ember ; Liu, Jinxu ; Dravid, Shashank M. ; Dingledine, Raymond ( February 2019 , Epilepsia)

   Seizures develop in 80% of patients with anti–N‐methyl‐d‐aspartate receptor (NMDAR) encephalitis, and these represent a major cause of morbidity and mortality. Anti‐NMDAR antibodies have been linked to memory loss in encephalitis; however, their role in seizures has not been established. We determined whether anti‐NMDAR antibodies from autoimmune encephalitis patients are pathogenic for seizures.

   We performed continuous intracerebroventricular infusion of cerebrospinal fluid (CSF) or purified immunoglobulin (IgG) from the CSF of patients with anti‐NMDAR encephalitis or polyclonal rabbit anti‐NMDAR IgG, in male C57BL/6 mice. Seizure status during a 2‐week treatment was assessed with video‐electroencephalography. We assessed memory, anxiety‐related behavior, and motor function at the end of treatment and assessed the extent of neuronal damage and gliosis in the CA1 region of hippocampus. We also performed whole‐cell patch recordings from the CA1 pyramidal neurons in hippocampal slices of mice with seizures.

   Prolonged exposure to rabbit anti‐NMDAR IgG, patient CSF, or human IgG purified from the CSF of patients with encephalitis induced seizures in 33 of 36 mice. The median number of seizures recorded in 2 weeks was 13, 39, and 35 per mouse in these groups, respectively. We observed only 18 brief nonconvulsive seizures in 11 of 29 control mice (median seizure count of 0) infused with vehicle (n = 4), normal CSF obtained from patients with noninflammatory central nervous system (CNS) conditions (n = 12), polyclonal rabbit IgG (n = 7), albumin (n = 3), and normal human IgG (n = 3). We did not observe memory deficits, anxiety‐related behavior, or motor impairment measured at 2 weeks in animals treated with CSF from affected patients or rabbit IgG. Furthermore, there was no evidence of hippocampal cell loss or astrocyte proliferation in the same mice.

   Our findings indicate that autoantibodies can induce seizures in anti‐NMDAR encephalitis and offer a model for testing novel therapies for refractory autoimmune seizures.

    

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4. Reactivity-dependent profiling of RNA 5-methylcytidine dioxygenases

   https://doi.org/10.1038/s41467-022-31876-2  

   Arguello, A. Emilia ; Li, Ang ; Sun, Xuemeng ; Eggert, Tanner W. ; Mairhofer, Elisabeth ; Kleiner, Ralph E. ( July 2022 , Nature Communications)

   Epitranscriptomic RNA modifications can regulate fundamental biological processes, but we lack approaches to map modification sites and probe writer enzymes. Here we present a chemoproteomic strategy to characterize RNA 5-methylcytidine (m⁵C) dioxygenase enzymes in their native context based upon metabolic labeling and activity-based crosslinking with 5-ethynylcytidine (5-EC). We profile m⁵C dioxygenases in human cells including ALKBH1 and TET2 and show that ALKBH1 is the major hm⁵C- and f⁵C-forming enzyme in RNA. Further, we map ALKBH1 modification sites transcriptome-wide using 5-EC-iCLIP and ARP-based sequencing to identify ALKBH1-dependent m⁵C oxidation in a variety of tRNAs and mRNAs and analyze ALKBH1 substrate specificity in vitro. We also apply targeted pyridine borane-mediated sequencing to measure f⁵C sites on select tRNA. Finally, we show that f⁵C at the wobble position of tRNA-Leu-CAA plays a role in decoding Leu codons under stress. Our work provides powerful chemical approaches for studying RNA m⁵C dioxygenases and mapping oxidative m⁵C modifications and reveals the existence of novel epitranscriptomic pathways for regulating RNA function.

    

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5. Nuclear‐encoded sigma factor 6 ( SIG 6) is involved in the block of greening response in Arabidopsis thaliana

   https://doi.org/10.1002/ajb2.1423  

   Alameldin, Hussien F. ; Oh, Sookyung ; Hernandez, Alexandra P. ; Montgomery, Beronda L. ( January 2020 , American Journal of Botany)

   Light is critical in the ability of plants to accumulate chlorophyll. When exposed to far‐red (FR) light and then grown in white light in the absence of sucrose, wild‐type seedlings fail to green in a response known as theFRblock of greening (BOG). This response is controlled by phytochrome A through repression of protochlorophyllide reductase‐encoding (POR) genes byFRlight coupled with irreversible plastid damage. Sigma (SIG) factors are nuclear‐encoded proteins that contribute to plant greening and plastid development through regulating gene transcription in chloroplasts and impacting retrograde signaling from the plastid to nucleus.SIGs are regulated by phytochromes, and the expression of someSIGfactors is reduced in phytochrome mutant lines, including phyA. Given the association of phyA with theFR BOGand its regulation ofSIGfactors, we investigated the potential regulatory role ofSIGfactors in theFR BOGresponse.

   We examinedFR BOGresponses insigmutants, phytochrome‐deficient lines, and mutant lines for several phy‐associated factors. We quantified chlorophyll levels and examined expression of keyBOG‐associated genes.

   Among sixsigmutants, only thesig6 mutant significantly accumulated chlorophyll afterFR BOGtreatment, similar to thephyAmutant.SIG6 appears to control protochlorophyllide accumulation by contributing to the regulation of tetrapyrrole biosynthesis associated with glutamyl‐tRNAreductase (HEMA1) function, select phytochrome‐interacting factor genes (PIF4andPIF6), andPENTA1, which regulatesPORAmRNAtranslation afterFRexposure.

   Regulation ofSIG6plays a significant role in plant responses toFRexposure during theBOGresponse.

    

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