Molecular Pathology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
EMBO J. 2010 Feb 17;29(4):749-60. doi: 10.1038/emboj.2009.397. Epub 2010 Jan 21.
Abundance of pseudo splice sites in introns can potentially give rise to innumerable pseudoexons, outnumbering the real ones. Nonetheless, these are efficiently ignored by the splicing machinery, a process yet to be understood completely. Although numerous 5' splice site-like sequences functioning as splicing silencers have been found to be enriched in predicted human pseudoexons, the lack of active pseudoexons pose a fundamental challenge to how these U1snRNP-binding sites function in splicing inhibition. Here, we address this issue by focusing on a previously described pathological ATM pseudoexon whose inhibition is mediated by U1snRNP binding at intronic splicing processing element (ISPE), composed of a consensus donor splice site. Spliceosomal complex assembly demonstrates inefficient A complex formation when ISPE is intact, implying U1snRNP-mediated unproductive U2snRNP recruitment. Furthermore, interaction of SF2/ASF with its motif seems to be dependent on RNA structure and U1snRNP interaction. Our results suggest a complex combinatorial interplay of RNA structure and trans-acting factors in determining the splicing outcome and contribute to understanding the intronic splicing code for the ATM pseudoexon.
内含子中大量的假剪接位点可能会产生无数的假外显子，数量超过真正的外显子。尽管如此，这些假外显子被剪接机制有效地忽略了，这个过程还没有被完全理解。虽然已经发现了许多类似于 5' 剪接位点的序列，作为剪接沉默子富集在预测的人类假外显子中，但缺乏活性的假外显子对这些 U1snRNP 结合位点在剪接抑制中的作用提出了一个基本的挑战。在这里，我们通过关注一个先前描述的病理性 ATM 假外显子来解决这个问题，该假外显子的抑制是由 U1snRNP 在由一致的供体位点组成的内含子剪接加工元件（ISPE）处结合介导的。剪接体复合物的组装表明，当 ISPE 完整时，A 复合物的形成效率低下，这意味着 U1snRNP 介导的非生产性 U2snRNP 的募集。此外，SF2/ASF 与其基序的相互作用似乎依赖于 RNA 结构和 U1snRNP 的相互作用。我们的结果表明，在决定剪接结果方面，RNA 结构和反式作用因子之间存在复杂的组合相互作用，并有助于理解 ATM 假外显子的内含子剪接密码。
