Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, USA.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, USA
RNA. 2024 Feb 16;30(3):240-255. doi: 10.1261/rna.079713.123.
XIST noncoding RNA promotes the initiation of X chromosome silencing by recruiting the protein SPEN to one X chromosome in female mammals. The SPEN protein is also called SHARP (SMRT and HDAC-associated repressor protein) and MINT (Msx-2 interacting nuclear target) in humans. SPEN recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We dissected the contributions of different RNA and protein regions to the formation of a human XIST-SPEN complex in vitro and identified novel sequence and structure determinants that may contribute to X chromosome silencing initiation. Binding of SPEN to XIST RNA requires RRM 4 of the protein, in contrast to the requirement of RRM 3 and RRM 4 for specific binding to SRA RNA. Measurements of SPEN binding to full-length, dimeric, trimeric, or other truncated versions of the A-repeat region revealed that high-affinity binding of XIST to SPEN in vitro requires a minimum of four A-repeat segments. SPEN binding to XIST A-repeat RNA changes the accessibility of the RNA at specific nucleotide sequences, as indicated by changes in RNA reactivity through chemical structure probing. Based on computational modeling, we found that inter-repeat duplexes formed by multiple A-repeats can present an unpaired adenosine in the context of a double-stranded region of RNA. The presence of this specific combination of sequence and structural motifs correlates with high-affinity SPEN binding in vitro. These data provide new information on the molecular basis of the XIST and SPEN interaction.
XIST 非编码 RNA 通过招募蛋白质 SPEN 到雌性哺乳动物的一条 X 染色体上，促进 X 染色体沉默的起始。SPEN 蛋白在人类中也被称为 SHARP（SMRT 和 HDAC 相关的抑制蛋白）和 MINT（Msx-2 相互作用的核靶标）。SPEN 招募 N-CoR2 和 HDAC3 启动 X 染色体上的组蛋白去乙酰化，导致抑制性染色质标记的形成和基因表达沉默。我们在体外剖析了不同 RNA 和蛋白质区域对人 XIST-SPEN 复合物形成的贡献，并确定了可能有助于 X 染色体沉默起始的新序列和结构决定因素。SPEN 与 XIST RNA 的结合需要蛋白质的 RRM4，与特定结合 SRA RNA 需要 RRM3 和 RRM4 形成对比。测量 SPEN 与全长、二聚体、三聚体或 A 重复区的其他截断版本的结合表明，XIST 在体外与 SPEN 的高亲和力结合需要至少四个 A 重复段。SPEN 与 XIST A 重复 RNA 的结合改变了 RNA 在特定核苷酸序列上的可及性，如通过化学结构探测改变 RNA 反应性所表明的。基于计算建模，我们发现多个 A 重复形成的重复间双链可以在 RNA 的双链区域中呈现未配对的腺苷。这种特定的序列和结构基序组合的存在与体外高亲和力 SPEN 结合相关。这些数据提供了 XIST 和 SPEN 相互作用的分子基础的新信息。
