Long Noncoding RNA LINC02453 Inhibits HIV‐1 Replication by Binding With SEC13 to Regulate the Viral Productive Cycle
Chen XiuRongfeng ChenLiufang WenTongxue QinYinlu LiaoXing TaoZongxiang YuanWudi WeiJinmiao LiYoujin HuangWenfei WeiJie LiuJinming SuYe LiHao LiangJunjun Jiang
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ABSTRACT Emerging evidence underscores the pivotal role of long noncoding RNAs (lncRNAs) as crucial regulators within the HIV life cycle. However, the precise functions and detailed mechanisms by which lncRNAs operate in HIV‐1 highly exposed but persistently seronegative (HESN) individuals remain currently unknown. Through RNA sequencing analysis of the HESN individual and the matched control, we identified potential lncRNAs. Then, we conducted validation experiments at the population level, while cellular models of HIV‐1 infection were constructed for functional experimental investigations in vitro. Subcellular localization of the identified lncRNA was determined, followed by an exploration of the specific regulatory mechanism underlying HIV resistance through some experiments, such as RNA pull‐down, western blot and Hirt assays. LncRNA LINC02453 is highly expressed in HESN. Moreover, LINC02453 is identified as a novel lncRNA associated with heightened resistance to HIV‐1. LINC02453 is predominantly localized in the nucleus and binds to SEC13, a component of the nuclear pore complex, leading to the inhibition of HIV‐1 replication by regulating key processes such as late reverse transcription, nuclear import, and DNA integration. Our findings suggest that LINC02453 may serve as a prospective target for the development of innovative anti‐HIV therapeutics.Keywords:
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Next generation sequencing (NGS) and especially ribonucleic acid (RNA) sequencing is a powerful tool to acquire insights into molecular disease mechanisms. Therefore, it is of interest to optimize methods for RNA extraction from archival, formalin fixed and paraffin embedded (FFPE) tissues. This is challenging due to RNA degradation and chemical modifications. The aim of this study was to find the most appropriate method to extract RNA from FFPE renal tissue to enable NGS.We evaluated seven commercially available RNA extraction kits: High Pure FFPE RNA Isolation (Roche), ExpressArt Clear FFPE RNAready (Amsbio), miRNeasy FFPE, RNeasy FFPE (Qiagen), PureLink FFPE Total RNA (Invitrogen), RecoverAll Total Nucleic Acid Isolation (Ambion) and Absolutely RNA FFPE Kit (Agilent). RNA was obtained from tissue blocks of two healthy, male Wistar rats and from normal renal tissue of patients undergoing nephrectomy. Yield and quality of RNA extracted from rat whole kidney sections, human kidney core biopsies and laser capture microdissected (LCM) glomerular cross-sections were assessed: Analyses of RNA quantity were performed using NanoDrop and Qubit. RNA quality is reflected by DV200 values (% of RNA fragments >200 nucleotides) utilizing the Agilent 2100 BioAnalyzer. RNA of human LCM samples was subsequently sequenced using the Illumina TruSeq(®) RNA Access Library Preparation Kit.Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.
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