Quantitative evaluation of all hexamers as exonic splicing elements
Quantitative evaluation of all hexamers as exonic splicing elements
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DOI：
10.1101/gr.119628.110
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发表时间：
2011-08-01
期刊：
影响因子：
7
通讯作者：
Chasin, Lawrence A.
中科院分区：
文献类型：
--
作者：
Ke, Shengdong;Shang, Shulian;Chasin, Lawrence A.
我们描述了通过对成功剪接的转录本进行深度测序来全面定量衡量一套完整的RNA6-mer序列的剪接影响。所有4096个6-MERS被替换在一个3-外显子微型基因的两个不同内部外显子内的5个位置，并对数百万个成功剪接的转录本进行了测序。结果允许为每个突变分子分配一个相对剪接强度分数。6-MERS对剪接的影响通常取决于它们的位置；这种上下文效应在很大程度上可以归因于在每个位点产生不同的重叠序列。考虑到这些重叠，每个6-聚体的剪接效应可以被量化，6-MERS可以被指定为增强剂(ESEseqs)和沉默剂(ESSseqs)，ESRseq分数表明它们的强度。一些6-MERS表现出相对于两个剪接点的位置偏差。这些ESRseq在人类外显子及其周围的分布和保守支持它们的分类。预测的RNA二级结构效应：有效的增强子、抑制子和3‘剪接位点倾向于单链，有效的5’剪接位点倾向于双链。6-MERS可能与另一种药物形成正或负的协同作用。染色质结构也可能影响所观察到的剪接增强，因为剪接性能与预测的6-MERS核小体占有率之间有很好的一致性。这种方法可能被证明在定义核酸调控基序方面具有普遍用途，在大多数情况下可以取代功能SELEX，并提供对剪接机制的见解。
We describe a comprehensive quantitative measure of the splicing impact of a complete set of RNA 6-mer sequences by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells. The results allowed the assignment of a relative splicing strength score to each mutant molecule. The effect of 6-mers on splicing often depended on their location; much of this context effect could be ascribed to the creation of different overlapping sequences at each site. Taking these overlaps into account, the splicing effect of each 6-mer could be quantified, and 6-mers could be designated as enhancers (ESEseqs) and silencers (ESSseqs), with an ESRseq score indicating their strength. Some 6-mers exhibited positional bias relative to the two splice sites. The distribution and conservation of these ESRseqs in and around human exons supported their classification. Predicted RNA secondary structure effects were also seen: Effective enhancers, silencers and 3' splice sites tend to be single stranded, and effective 5' splice sites tend to be double stranded. 6-mers that may form positive or negative synergy with another were also identified. Chromatin structure may also influence the splicing enhancement observed, as a good correspondence was found between splicing performance and the predicted nucleosome occupancy scores of 6-mers. This approach may prove of general use in defining nucleic acid regulatory motifs, substitute for functional SELEX in most cases, and provide insights about splicing mechanisms.