A multiprotein occupancy map of the mRNP on the 3' end of histone mRNAs.
A multiprotein occupancy map of the mRNP on the 3' end of histone mRNAs.
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DOI：
10.1261/rna.053389.115
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发表时间：
2015-11
期刊：
影响因子：
--
通讯作者：
Whitfield ML
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文献类型：
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作者：
Brooks L 3rd;Lyons SM;Mahoney JM;Welch JD;Liu Z;Marzluff WF;Whitfield ML
动物复制依赖性 (RD) 组蛋白 mRNA 与染色体复制协调调节。 RD-组蛋白 mRNA 是唯一已知的未聚腺苷酸化的细胞 mRNA。相反，成熟的转录本以保守的茎环（SL）结构结束。该 SL 结构与茎环结合蛋白 (SLBP) 相互作用，后者参与 RD-组蛋白 mRNA 代谢的各个方面。我们使用了多种基因组方法，包括交联免疫沉淀物高通量测序 (HITS-CLIP) 来分析 SLBP 的 RNA 结合情况。 SLBP 不与组蛋白 mRNA 以外的任何 RNA 结合。我们对 HITS-CLIP 数据进行了生物信息学分析，包括 (i) 使用 CVCA 通过测序读取覆盖率对基因进行聚类，(ii) 绘制结合的 RNA 片段末端图，以及 (iii) 使用 CLIP-PyL 软件绘制交联诱导突变位点 (CIMS)。这些分析使我们能够确定 SLBP 与其 RD-组蛋白 mRNA 配体之间分子接触的特定位点。我们进行了体外交联测定以完善 CIMS 图谱，发现组蛋白 mRNA SL 环中的尿嘧啶一和三优先与 SLBP 交联，而环中的尿嘧啶二优先与单独的组件（可能是 3'hExo）交联。我们还对 iCLIP 数据集进行了二次分析，以绘制 RD-组蛋白 mRNA 上的 UPF1 占据情况，发现 UPF1 与 SLBP 结合位点相邻。多种蛋白质可能将 RD-组蛋白 mRNA 的 3' 端与 SLBP 结合在一起。
The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem–loop (SL) structure. This SL structure interacts with the stem–loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3′hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3′ end of RD-histone mRNAs together with SLBP.