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Molecular Characterization of LncRNAs: ANRIL as a Model System

Abstract : The increased knowledge on long non-coding RNA (lncRNA ≥ 200-nts lacking obvious coding regions) and their involvement in global genome regulation push the lncRNA-disease interconnection to a promising and timely field of research. Due to flexible 2D structures and multiple protein binding sites, lncRNAs carry intrinsic features conferring a wide regulatory potential. Although lncRNAs are being extensively studied, the field is still suffering from a lack of knowledge on their structure/function relationships at the molecular and cellular levels. The team focuses on the characterization of the lncRNA named ANRIL (Antisense Non-coding RNA in the INK4 Locus). ANRIL is an ideal study model since it is associated to human diseases and is likely implicated in regulating the fine-tuning of gene expression via the recruitment of nucleoprotein complexes. Until now, ANRIL functions have only been superficially explored at the molecular level. ANRIL belongs to the 9p21 locus that is silenced by ANRIL via the recruitment of the polycomb repressor complex PRC1 and PRC2. Even though the trans-regulatory activity of ANRIL was demonstrated by previous studies, its molecular aspect needed to be refined. For instance, the coding and the non-coding genes contacted and regulated by ANRIL in a direct manner remained unknown. Also not documented were the mechanisms engaged by ANRIL to associate specifically with the genome. Our work tends to refine the so-called trans-activity by identifying the genes directly contacted and regulated by ANRIL. We identified genome-wide the chromatin occupancy of ANRIL in HEK293 by applying the ChIRP-seq approach and found that ANRIL associates with 3227 binding sites mostly composed by G/A residues. By crossing the ChIRP-seq with transcriptomic data from ANRIL knocked-down cells, we established a list of 189 genes corresponding to the primary trans-targets of ANRIL since they were both contacted and affected by the presence of ANRIL. Among them, 123 genes were found to be negatively regulated by ANRIL. In silico approaches highlighted the presence of multiple classes of transposable elements throughout ANRIL exons. In particular, 70% of the longest Exon8 was made up of ERVL element. We investigated its putative role in ANRIL's trans-activity. We showed that it is at least partially responsible for the association of ANRIL to the chromatin since deletion of Exon8 resulted in a severe reduction of ANRIL's genomic occupancy. By applying highly stringent criteria, we accurately identified 9 out of the 123 trans-target genes of ANRIL which expression specifically depends on Exon8. By further in silico, in cellulo and in vitro characterization, we showed that Exon8 contains a 42-nts sequence contributing in the recognition and subsequently in the silencing of the FIRRE and TPD52L1 genes. We brought evidences in favor of a recognition mode involving DNA/DNA:RNA triplex formation. Furthermore, we initiated the identification of ANRIL’s protein partners. By in vitro and in cellulo approaches, we identified hnRNP K as a novel partner of ANRIL. We evidenced a potential coregulatory role of ANRIL and hnRNP K on the 9p21 locus and other distal inflammatory loci. Finally, using the Stanislas Cohort, the identification of RNA and DNA biomarkers for the early diagnosis of cardiovascular and metabolic diseases has been initiated. GWAS data revealed novel associations of 9p21 locus SNPs with cardiovascular and metabolic diseases. We think that the conjugation of cellular, molecular and biochemical approaches we deployed in the study allowed to generate a clearer vision of ANRIL activity and its molecular links to diseases.
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Submitted on : Thursday, September 17, 2020 - 2:56:19 PM
Last modification on : Tuesday, June 21, 2022 - 11:35:11 AM

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  • HAL Id : tel-02941984, version 1


Charbel Alfeghaly. Molecular Characterization of LncRNAs: ANRIL as a Model System. Genomics [q-bio.GN]. Université de Lorraine, 2020. English. ⟨NNT : 2020LORR0054⟩. ⟨tel-02941984⟩



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