A long noncoding RNA antagonizes the PBAF complex to promote ccRCC progression

项目概述：我们发现了一种完全未表征的长非编码RNA（lncRNA）ADIRF- AS 1与肿瘤抑制因子多溴和BRG 1相关因子（PBAF）染色质结合 修饰复合物，包括PBAF复合物的三种独特的蛋白质组分：ARID 2，BRD 7，和 PBRM 1。含有PBAF复合物布罗莫结构域的蛋白PBRM 1在约40%的透明细胞中突变。 细胞肾癌（ccRCC），其中PBRM 1的缺失增加增殖和总体肿瘤负荷。公开 现有的患者数据显示，与正常人相比，ADIRF-AS 1在ccRCC肿瘤中的表达更高， 组织中我们在一组ccRCC细胞系中过表达ADIRF-AS 1，发现ADIRF-AS 1过表达 仅在具有野生型PBAF蛋白表达的ccRCC细胞系中显著促进增殖 件.重要的是，我们发现ADIRF-AS 1的过表达降低了PBAF的蛋白表达， 蛋白质成分接下来，我们试图确定ADIRF-AS 1如何负调控PBAF复合物。我们 进行了lncRNA下拉分析，然后进行了蛋白质组学分析，发现沿着 PBAF复合物ADIRF-AS 1还与E3泛素连接酶TRIM 25结合，已显示TRIM 25 靶向PBRM 1用于蛋白酶体降解。我们证实沉默TRIM 25增加PBRM 1表达， 并发现蛋白酶体抑制剂挽救了ADIRF-AS 1后PBRM 1表达的下降， 过度表达因此，我假设ADIRF-AS 1与PBAF复合物相互作用，以促进其表达。 通过充当PBRM 1和TRIM 25的支架来降解，促进肿瘤形成和进展。 为了解决这一假设，我们提出了两个目标：（1）确定ADIRF-AS 1表达在PBAF中的作用 介导的代谢和肿瘤发生，以及（2）表征ADIRF-AS 1负性介导的代谢和肿瘤发生的机制， 通过TRIM 25调节PBAF复合物以促进肿瘤发生。

Project Summary: We discovered that a completely uncharacterized long noncoding RNA (lncRNA) ADIRF- AS1 binds to the tumor suppressor Polybromo- and BRG1-associated factors-containing (PBAF) chromatin modifying complex, including the three unique protein components of the PBAF complex: ARID2, BRD7, and PBRM1. The PBAF complex bromodomain containing protein PBRM1 is mutated in approximately 40% of clear cell renal carcinoma (ccRCC), where loss of PBRM1 increases proliferation and overall tumor burden. Publicly available patient data revealed that ADIRF-AS1 is more highly expressed in ccRCC tumors compared to normal tissues. We overexpressed ADIRF-AS1 in a panel of ccRCC cell lines and found that ADIRF-AS1 overexpression significantly promoted proliferation only in ccRCC cell lines with wildtype expression of PBAF protein components. Importantly, we found that overexpression of ADIRF-AS1 decreased protein expression of PBAF protein components. Next, we sought to identify how ADIRF-AS1 negatively regulates the PBAF complex. We performed a lncRNA pulldown assay followed by proteomics analysis and found that along with components of the PBAF complex, ADIRF-AS1 also associates with an E3 ubiquitin ligase TRIM25, which has been shown to target PBRM1 for proteasomal degradation. We validated that silencing TRIM25 increases PBRM1 expression and found that proteasome inhibitors rescued decreased expression of PBRM1 after ADIRF-AS1 overexpression. Therefore, I hypothesize that ADIRF-AS1 interacts with the PBAF complex to promote its degradation by acting as a scaffold for PBRM1 and TRIM25, promoting tumor formation and progression. To address this hypothesis, we propose two aims to: (1) Determine the role of ADIRF-AS1 expression in PBAF mediated metabolism and tumorigenesis, and (2) Characterize the mechanism by which ADIRF-AS1 negatively regulates the PBAF complex through TRIM25 to promote tumorigenesis.