Molecular Cloning and Expression Analysis of a Putative Nuclear Protein, SR-25

We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as “SR-25.” In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

An attempt to detect siRNA-mediated genomic DNA modification by artificially induced mismatch siRNA in Arabidopsis.

Although tremendous progress has been made in recent years in identifying molecular mechanisms of small interfering RNA (siRNA) functions in higher plants, the possibility of direct interaction between genomic DNA and siRNA remains an enigma. Such an interaction was proposed in the 'RNA cache' hypothesis, in which a mutant allele is restored based on template-directed gene conversion. To test this hypothesis, we generated transgenic Arabidopsis thaliana plants conditionally expressing a hairpin dsRNA construct of a mutated acetolactate synthase (mALS) gene coding sequence, which confers chlorsulfuron resistance, in the presence of dexamethasone (DEX). In the transgenic plants, suppression of the endogenous ALS mRNA expression as well as 21-nt mALS siRNA expression was detected after DEX treatment. After screening >100,000 progeny of the mALS siRNA-induced plants, no chlorsulfuron-resistant progeny were obtained. Further experiments using transgenic calli also showed that DEX-induced expression of mALS siRNA did not affect the number of chlorsulfuron-resistant calli. No trace of cytosine methylation of the genomic ALS region corresponding to the dsRNA region was observed in the DEX-treated calli. These results do not necessarily disprove the 'RNA cache' hypothesis, but indicate that an RNAi machinery for ALS mRNA suppression does not alter the ALS locus, either genetically or epigenetically

Phenotypes of transgenic <i>Arabidopsis</i> plants expressing <i>mALS</i> siRNA after DEX treatment.

<p>(A) Beta-glucuronidase (GUS) expression in root tissues of the transgenic plant lines #3, #6 and #12 after treatment with (+) or without (-) DEX. (B) Relative mRNA expression of the <i>ALS</i> gene in the wild-type (WT) and transgenic (#6) plants with or without (-) DEX treatment. <i>ALS</i> mRNA expression was normalized to the expression level of <i>Act8</i>. Experiments were replicated three times. (C) Expression of siRNA derived from <i>mALS</i> dsRNA in the transgenic plants (lines #3, #4, #6 and #12) after DEX treatment. DEX was applied for 2 days. Ethidium bromide staining of tRNA is shown as a loading control. (D) Effect on <i>mALS</i> siRNA expression of subculturing onto medium containing DEX in the #6 transgenic plants. Plants cultured after one (S1) to three (S3) rounds of subculturing or without subculturing (no S) for different periods (indicated as days after treatment) were analyzed.</p

Selection of chlorsulfuron-resistant selfed progeny of the transgenic plants treated with or without DEX.

<p>Seeds were sown on MS medium containing 100 nM chlorsulfuron. CS^S, number of chlorsulfuron-sensitive seedlings. CS^R, number of chlorsulfuron-resistant seedlings.</p

Schematic diagram of mutated acetolactate synthase (<i>mALS</i>) dsRNA induction vector and possible consequence of endogenous <i>ALS</i> mRNA degradation and hypothetical modification of the genomic <i>ALS</i> sequence.

<p>The <i>mALS</i> dsRNA is transcribed from the pOpOff2mALSir binary vector (upper) after dexamethasone (DEX) treatment, which activates LhGR transcription factor targeting the pOp6 bidirectional promoter. The <i>mALS</i> dsRNA is processed into siRNAs, which in turn compose an RNA-induced silencing complex (RISC) and the endogenous <i>ALS</i> mRNA is expected to be degraded by the RNAi machinery. At the same time, accumulation of <i>mALS</i> siRNA may be guided to the complementary genomic <i>ALS</i> locus (lower), resulting in <i>ALS</i> mutation, which would confer chlorsulfuron resistance. The black circles on the <i>ALS</i> locus represent hypothetical DNA methylation caused by RNA-dependent DNA methylation (RdDM).</p