Drosha promotes splicing of a pre-microRNA-like alternative exon.
Drosha promotes splicing of a pre-microRNA-like alternative exon.
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DOI：
10.1371/journal.pgen.1004312
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发表时间：
2014-05
期刊：
影响因子：
4.5
通讯作者：
Hastings ML
中科院分区：
文献类型：
--
作者：
Havens MA;Reich AA;Hastings ML
核糖核酸酶 III 酶 Drosha 通过将初级 RNA 转录物中的发夹结构结合和切割为前体 miRNA (pre-miRNA)，在 microRNA (miRNA) 的生物发生中发挥核心作用。许多 miRNA 基因位于蛋白质编码宿主基因内，并被 Drosha 切割，其切割方式与剪接体对内含子的剪接一致。剪接和 pre-miRNA 生物发生的密切关系表明 miRNA 和宿主基因表达具有共同调节的潜力，尽管这种关系尚未完全了解。在这里，我们描述了 Drosha 在外显子剪接中的独立于切割的作用，该外显子具有类似于 Drosha 底物的预测发夹结构。我们发现 Drosha 可以在体外和细胞内将 eIF4H 基因的选择性剪接外显子 5 切割成 pre-miRNA。然而，Drosha 在 eIF4H 基因表达中的主要作用是促进外显子 5 的剪接。Drosha 与外显子结合并以依赖于 RNA 结构但不依赖于 Drosha 切割的方式增强剪接。我们得出结论，Drosha 可以像剪接增强子一样发挥作用并促进外显子包含。我们的结果揭示了一种新的选择性剪接调节机制，涉及 Drosha 在剪接中的独立于切割的作用。 MicroRNA (miRNA) 是短的非编码 RNA，在基因沉默中发挥作用，是通过微处理器执行的反应从较大的初级 RNA 转录物中裂解而产生的。初级 miRNA 转录物通常位于基因的内含子内。因此，微处理器和负责前 mRNA 剪接的剪接体都与相同的序列相互作用，尽管人们对这两个过程如何相互影响知之甚少。在这项研究中，我们发现选择性剪接的 eIF4H 外显子 5 预计会形成类似于微处理器底物的 RNA 发夹。我们发现微处理器可以结合并切割外显子 5，从而阻止该外显子包含在 mRNA 中。然而，我们发现 Drosha（微处理器的一个组件）的主要功能是在体外和细胞内增强外显子 5 剪接，而不是切割 RNA。我们的结果表明微处理器在剪接中的作用不同于其在 miRNA 生物发生中的作用。这种微处理器的活动代表了复合体的一种新功能，可能是调节选择性剪接的重要机制。
The ribonuclease III enzyme Drosha has a central role in the biogenesis of microRNA (miRNA) by binding and cleaving hairpin structures in primary RNA transcripts into precursor miRNAs (pre-miRNAs). Many miRNA genes are located within protein-coding host genes and cleaved by Drosha in a manner that is coincident with splicing of introns by the spliceosome. The close proximity of splicing and pre-miRNA biogenesis suggests a potential for co-regulation of miRNA and host gene expression, though this relationship is not completely understood. Here, we describe a cleavage-independent role for Drosha in the splicing of an exon that has a predicted hairpin structure resembling a Drosha substrate. We find that Drosha can cleave the alternatively spliced exon 5 of the eIF4H gene into a pre-miRNA both in vitro and in cells. However, the primary role of Drosha in eIF4H gene expression is to promote the splicing of exon 5. Drosha binds to the exon and enhances splicing in a manner that depends on RNA structure but not on cleavage by Drosha. We conclude that Drosha can function like a splicing enhancer and promote exon inclusion. Our results reveal a new mechanism of alternative splicing regulation involving a cleavage-independent role for Drosha in splicing. MicroRNAs (miRNAs) are short non-coding RNAs that function in gene silencing and are produced by cleavage from a larger primary RNA transcript through a reaction that is carried out by the Microprocessor. Primary miRNA transcripts are often located within the introns of genes. Thus, both the Microprocessor and the spliceosome, which is responsible for pre-mRNA splicing, interact with the same sequences, though little is known about how these two processes influence each other. In this study, we discovered that the alternatively spliced eIF4H exon 5 is predicted to form an RNA hairpin that resembles a Microprocessor substrate. We found that the Microprocessor can bind and cleave exon 5, which precludes inclusion of the exon in the mRNA. However, we find that Drosha, a component of the Microprocessor, primarily functions to enhance exon 5 splicing both in vitro and in cells, rather than to cleave the RNA. Our results suggest that the Microprocessor has a role in splicing that is distinct from its role in miRNA biogenesis. This Microprocessor activity represents a new function for the complex that may be an important mechanism for regulating alternative splicing.