Abstract

A large portion of the human genome is transcribed into long noncoding RNAs that can range

from 200 nucleotides to several kilobases in length. The number of identified lncRNAs is still

growing, but only a handful of them have been functionally characterized. However, it is known

that the functions of lncRNAs are closely related to their subcellular localization. Cytoplasmic lncRNAs can regulate mRNA stability, affect translation and act as miRNA sponges, while nuclear-retained long noncoding RNAs have been reported to be involved in transcriptional control, chromosome scaffolding, modulation of alternative splicing and chromatin remodelling. Through these processes, lncRNAs have diverse regulatory roles in cell biology and diseases. OIP5-AS1 (also known as Cyrano), a poorly characterized lncRNA expressed antisense to the OIP5 oncogene, is deregulated in multiple cancers. We showed that one of the OIP5-AS1 splicing forms (ENST00000501665.2) is retained in the cell nucleus where it associates with chromatin, thus narrowing down the spectrum of its possible mechanisms of action. Its knockdown with antisense LNA gapmeRs led to inhibited expression of a sense partner, OIP5, strongly suggesting a functional coupling between OIP5 and ENST00000501665.2. A subsequent bioinformatics analysis followed by RAP-MS and RNA Immunoprecipitation experiments suggested its possible mode of action; in particular, we found that ENST00000501665.2 directly binds to a number of nuclear proteins, including SMARCA4, a component of the SWI/SNF chromatin remodelling complex, whose binding motif is located in the promoter of the OIP5 oncogene.

摘要

人类基因组的很大一部分被转录为长的非编码RNA

从200个核苷酸到几千个碱基的长度。鉴定出的lncRNA的数量仍在

增长，但其中只有少数具有功能特征。但是，众所周知

lncRNA的功能与其亚细胞定位密切相关。细胞质lncRNA可以调节mRNA稳定性，影响翻译并充当miRNA海绵，而据报道，核保留的长非编码RNA参与转录控制，染色体支架，其他剪接的调节和染色质重塑。通过这些过程，lncRNA在细胞生物学和疾病中具有多种调节作用。OIP5-AS1（也称为Cyrano），一种表达不佳的，与OIP5癌基因反义的lncRNA ，在多种癌症中均受到抑制。我们证明了OIP5-AS1之一剪接形式（ENST00000501665.2）被保留在细胞核中，与染色质结合，从而缩小了其可能的作用机理的范围。其与反义LNA gapmeR的组合导致有义伴侣OIP5的表达受到抑制，强烈暗示了OIP5与ENST0000050165.2之间的功能偶联。随后的生物信息学分析，然后进行RAP-MS和RNA免疫沉淀实验，表明其可能的作用方式。特别是，我们发现ENST00000501665.2直接与许多核蛋白结合，包括SMARCA4（SWI / SNF染色质重塑复合体的一个组成部分），其结合基序位于OIP5癌基因的启动子中。