Alternative 3′-end processing of long noncoding RNA initiates construction of nuclear paraspeckles
Alternative 3′-end processing of long noncoding RNA initiates construction of nuclear paraspeckles
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DOI：
10.1038/emboj.2012.251
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发表时间：
2012-10-17
期刊：
影响因子：
11.4
通讯作者：
Hirose, Tetsuro
中科院分区：
文献类型：
--
作者：
Naganuma, Takao;Nakagawa, Shinichi;Hirose, Tetsuro
Paraspeckles 是围绕特定长非编码 RNA NEAT1 构建的独特亚核结构，NEAT1 由通过替代 3' 端加工产生的两种亚型（NEAT1_1 和 NEAT1_2）组成。为了解决导致副斑点形成的精确分子过程，我们主要通过与荧光蛋白标记的全长 cDNA 文库的共定位筛选，鉴定了 35 种副斑点蛋白 (PSP)。大多数新发现的 PSP 具有各种假定的 RNA 结合结构域。随后的 RNAi 分析确定了副斑纹形成所必需的 7 个 PSP。重要的 PSP 之一 HNRNPK 似乎通过负向调节 NEAT1_1 同工型的 3' 端多腺苷酸化来影响重要的 NEAT1_2 同工型的产生。体外 3' 末端加工测定表明，HNRNPK 在 NEAT1_1 的替代多聚腺苷酸化位点附近阻止了 CPSF6-NUDT21 (CFIm) 复合物的结合。体外结合测定表明，HNRNPK 与 CPSF6 竞争与 NUDT21 的结合，这是 HNRNPK 阻止 CFIm 结合的潜在机制。 HNRNPK 功能导致 NEAT1_2 的优先积累，并启动多个 PSP 的副散斑构建。 EMBO 杂志 (2012) 31, 4020-4034。 doi：10.1038/emboj.2012.251； 2012 年 9 月 7 日在线发布
Paraspeckles are unique subnuclear structures built around a specific long noncoding RNA, NEAT1, which is comprised of two isoforms produced by alternative 3'-end processing (NEAT1_1 and NEAT1_2). To address the precise molecular processes that lead to paraspeckle formation, we identified 35 paraspeckle proteins (PSPs), mainly by colocalization screening with a fluorescent protein-tagged full-length cDNA library. Most of the newly identified PSPs possessed various putative RNA-binding domains. Subsequent RNAi analyses identified seven essential PSPs for paraspeckle formation. One of the essential PSPs, HNRNPK, appeared to affect the production of the essential NEAT1_2 isoform by negatively regulating the 3'-end polyadenylation of the NEAT1_1 isoform. An in vitro 3'-end processing assay revealed that HNRNPK arrested binding of the CPSF6-NUDT21 (CFIm) complex in the vicinity of the alternative polyadenylation site of NEAT1_1. In vitro binding assays showed that HNRNPK competed with CPSF6 for binding to NUDT21, which was the underlying mechanism to arrest CFIm binding by HNRNPK. This HNRNPK function led to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction with multiple PSPs. The EMBO Journal (2012) 31, 4020-4034. doi:10.1038/emboj.2012.251; Published online 7 September 2012