Department of Developmental Biology, Sloan-Kettering Institute, New York, NY, USA.
Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Nat Struct Mol Biol. 2018 May;25(5):365-371. doi: 10.1038/s41594-018-0052-6. Epub 2018 Apr 9.
Many long Drosophila introns are processed by an unusual recursive strategy. The presence of ~200 adjacent splice acceptor and splice donor sites, termed ratchet points (RPs), were inferred to reflect 'zero-nucleotide exons', whose sequential processing subdivides removal of long host introns. We used CRISPR-Cas9 to disrupt several intronic RPs in Drosophila melanogaster, some of which recapitulated characteristic loss-of-function phenotypes. Unexpectedly, selective disruption of RP splice donors revealed constitutive retention of unannotated short exons. Assays using functional minigenes confirm that unannotated cryptic splice donor sites are critical for recognition of intronic RPs, demonstrating that recursive splicing involves the recognition of cryptic RP exons. This appears to be a general mechanism, because canonical, conserved splice donors are specifically enriched in a 40-80-nt window downstream of known and newly annotated intronic RPs and exhibit similar properties to a broadly expanded class of expressed RP exons. Overall, these studies unify the mechanism of Drosophila recursive splicing with that in mammals.
许多长的果蝇内含子是通过一种不寻常的递归策略进行加工的。存在大约 200 个相邻的剪接受体和剪接供体位点，称为棘轮点（RP），这被推断反映了“零核苷酸外显子”，其顺序加工将长的宿主内含子的去除进行细分。我们使用 CRISPR-Cas9 破坏了果蝇中的几个内含子 RP，其中一些再现了典型的功能丧失表型。出乎意料的是，选择性破坏 RP 剪接受体揭示了未注释的短外显子的组成性保留。使用功能性小基因进行的测定证实，未注释的隐性剪接供体位点对于识别内含子 RP 至关重要，这表明递归剪接涉及对隐性 RP 外显子的识别。这似乎是一种普遍的机制，因为规范的、保守的剪接受体在已知和新注释的内含子 RP 的下游 40-80nt 窗口中特异性富集，并表现出与广泛扩展的表达 RP 外显子类相似的特性。总的来说，这些研究将果蝇递归剪接的机制与哺乳动物的机制统一起来。
