Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast.
Transcription of lncRNA prt, clustered prt RNA sites for Mmi1 binding, and RNA polymerase II CTD phospho-sites govern the repression of pho1 gene expression under phosphate-replete conditions in fission yeast.
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DOI：
10.1261/rna.056515.116
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发表时间：
2016-07
期刊：
影响因子：
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通讯作者：
Schwer B
中科院分区：
文献类型：
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作者：
Chatterjee D;Sanchez AM;Goldgur Y;Shuman S;Schwer B
裂殖酵母 Pho1 酸性磷酸酶的表达在富含磷酸盐的培养基中生长期间受到抑制。抑制是通过 pho1 上游 prt 位点的转录介导的，以产生长非编码 (lnc) prt RNA。抑制还受 RNA 聚合酶 II CTD 磷酸化状态的控制，因此无法放置 Ser7-PO4 标记（如 S7A 中）会抑制 Pho1 表达，而无法放置 Thr4-PO4 标记（如 T4A 中）会在磷酸盐充足的细胞中过度抑制 Pho1。在这里，我们发现 prt-pho1 位点的基础 pho1 表达与 prt 启动子的活性呈负相关，prt 启动子位于 prt 转录起始位点之前的 110 个核苷酸的 DNA 片段中。 CTD 突变 S7A 和 T4A 对 prt 启动子或 pho1 启动子的活性没有影响，这表明 S7A 和 T4A 影响 prt lncRNA 合成的起始后事件，从而分别使其对 pho1 的抑制减弱和增强。 prt lncRNA 包含由含有 YTH 结构域的蛋白 Mmi1 识别的 DSR（选择性去除决定因素）序列簇。改变 prt lncRNA 中两个 DSR 簇的核碱基序列会导致磷酸盐充足的细胞中 pho1 的过度抑制，同时 prt 转录物水平增加。分离的 Mmi1 YTH 结构域与具有单个或串联 DSR 元件的 RNA 结合，并以非合作方式与后者结合。我们报告了 Mmi1 YTH 结构域的 1.75 Å 晶体结构，并提供了 Mmi1 通过与识别 m6A 修饰 RNA 的结构同源 YTH 蛋白不同的结合模式识别 DSR RNA 的证据。
Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt–pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m6A-modified RNA.