Intron splicing is a key regulatory step in gene expression in eukaryotes. Three sequence elements required for splicing—5′ and 3′ splice sites and a branchpoint—are especially well-characterized in Saccharomyces cerevisiae, but our understanding of additional intron features that impact splicing in this organism is incomplete, due largely to its small number of introns. To overcome this limitation, we constructed a library in S. cerevisiae of random 50-nt (N50) elements individually inserted into the intron of a reporter gene and quantified canonical splicing and the use of cryptic splice sites by sequencing analysis. More than 70% of approximately 140,000 N50 elements reduced splicing by at least 20%. N50 features, including higher GC content, presence of GU repeats, and stronger predicted secondary structure of its pre-mRNA, correlated with reduced splicing efficiency. A likely basis for the reduced splicing of such a large proportion of variants is the formation of RNA structures that pair N50 bases—such as the GU repeats—with other bases specifically within the reporter pre-mRNA analyzed. However, multiple models were unable to explain more than a small fraction of the variance in splicing efficiency across the library, suggesting that complex nonlinear interactions in RNA structures are not accurately captured by RNA structure prediction methods. Our results imply that the specific context of a pre-mRNA may determine the bases allowable in an intron to prevent secondary structures that reduce splicing. This large data set can serve as a resource for further exploration of splicing mechanisms.

中文翻译：

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插入内含子的随机 50 个碱基序列对酿酒酵母剪接的影响

内含子剪接是真核生物基因表达的关键调控步骤。剪接所需的三个序列元件（5' 和 3' 剪接位点以及分支点）在 酿酒酵母 中得到了很好的表征，但我们对其他序列元件的理解影响该生物体剪接的内含子特征是不完整的，这主要是由于其内含子数量较少。为了克服这个限制，我们在 S 中构建了一个库。酿酒酵母随机50-nt (N50)元件分别插入报告基因的内含子，并通过测序分析量化规范剪接和隐秘剪接位点的使用。大约 140,000 个 N50 元件中，超过 70% 的拼接减少了至少 20%。 N50 的特征，包括较高的 GC 含量、GU 重复的存在以及其前 mRNA 的更强的预测二级结构，与剪接效率降低相关。如此大比例的变体剪接减少的一个可能基础是RNA结构的形成，该结构将N50碱基（例如GU重复序列）与特别是在所分析的报告基因前体mRNA内的其他碱基配对。然而，多个模型无法解释整个文库中剪接效率差异的一小部分，这表明 RNA 结构预测方法无法准确捕获 RNA 结构中复杂的非线性相互作用。我们的结果表明，前体 mRNA 的特定背景可能决定内含子中允许的碱基，以防止减少剪接的二级结构。这个大数据集可以作为进一步探索拼接机制的资源。