In rice splice variants that restore the reading frame after frameshifting indel introduction are common, often induced by the indels and sometimes lead to organism-level rescue.
In rice splice variants that restore the reading frame after frameshifting indel introduction are common, often induced by the indels and sometimes lead to organism-level rescue.
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在水稻中，移码插入缺失后恢复阅读框的剪接变体很常见，通常由插入缺失诱导，有时会导致生物体水平的拯救
DOI：
10.1371/journal.pgen.1010071
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发表时间：
2022-03
期刊：
影响因子：
4.5
通讯作者：
Hurst LD
中科院分区：
文献类型：
--
作者：
Jia Y;Qin C;Traw MB;Chen X;He Y;Kai J;Yang S;Wang L;Hurst LD
移码非3 n插入缺失的引入使得能够鉴定基因-性状关联。然而，已经假设由于使用非规范剪接形式而恢复原始阅读框架可能导致拯救。到目前为止，很少有证据表明生物体水平的救援，这种机制，它是未知的，如何普遍indel诱导，或以其他方式与，框架恢复剪接形式。我们对水稻中随机选择的基因座进行CRISPR/Cas9编辑来调查这些问题。我们发现，大多数位点有一个框架恢复异构体。重要的是，这些同种型中的四分之三在不存在indel的情况下未观察到，这与indel通常诱导新的同种型一致。这一点得到了NMD被摧毁背景下的分析的支持。我们详细考虑了两个顶级救援候选人，在缺蜡花药1（wda 1）和脆秆（bc 10），发现在这两种情况下，生物体水平的救援是强大的，但由于不同的剪接修饰路线。然而，更普遍的是，由于框架恢复异构体的丰度低，而且可能具有太大的破坏性，我们认为这种拯救是罕见的例外，而不是规则。尽管如此，假设插入缺失通常诱导框架恢复亚型，这些结果强调需要检查RNA水平的影响，非3 n插入缺失，并建议在任何给定的基因中的多个非3 n插入缺失是可取的，以探测基因的性状协会。由于蛋白质编码基因是以三个为单位（密码子）读取的，因此预期长度不是三的倍数（非3 n）的插入或缺失（插入缺失）特别有害。它们是否是重要的，无论是解释非3 n插入缺失实验的结果，以探测基因的功能重要性和诊断。特别神秘的是发生在基因中的一些非3 n变化损害表型，而其他看似相似的却没有。对于后者的一种解释是，非3 n插入缺失可能通过框架恢复剪接形式被拯救。在这里，我们通过在水稻的许多基因中诱导非3 n插入/缺失来检验这一假设，发现许多非3 n插入/缺失与恢复阅读框架的剪接形式相关。在大多数这些情况下，插入缺失似乎诱导潜在的拯救剪接形式。我们详细研究了两个热门案例，并通过剪接修饰显示了功能性拯救。然而，更一般地，框架恢复形式是低丰度的，并且可能导致受损的蛋白质。我们的结论是，剪接介导的救援是可能的，但可能不常见。然而，在实验设计和解释中不应忽视这一点。
The introduction of frameshifting non-3n indels enables the identification of gene-trait associations. However, it has been hypothesised that recovery of the original reading frame owing to usage of non-canonical splice forms could cause rescue. To date there is very little evidence for organism-level rescue by such a mechanism and it is unknown how commonly indels induce, or are otherwise associated with, frame-restoring splice forms. We perform CRISPR/Cas9 editing of randomly selected loci in rice to investigate these issues. We find that the majority of loci have a frame-restoring isoform. Importantly, three quarters of these isoforms are not seen in the absence of the indels, consistent with indels commonly inducing novel isoforms. This is supported by analysis in the context of NMD knockdowns. We consider in detail the two top rescue candidates, in wax deficient anther 1 (wda1) and brittle culm (bc10), finding that organismal-level rescue in both cases is strong but owing to different splice modification routes. More generally, however, as frame-restoring isoforms are low abundance and possibly too disruptive, such rescue we suggest to be the rare exception, not the rule. Nonetheless, assuming that indels commonly induce frame-restoring isoforms, these results emphasize the need to examine RNA level effects of non-3n indels and suggest that multiple non-3n indels in any given gene are advisable to probe a gene’s trait associations. As protein coding genes are read in units of three (codons), insertions or deletions (indels) that are not a multiple of three long (non 3n) are expected to be especially harmful. Whether they are is important both for interpreting the results of non-3n indel experiments to probe a gene’s functional importance and for diagnostics. Particularly enigmatic are incidences where some non-3n changes in a gene compromise phenotypes while other seemingly comparable ones do not. One explanation for the latter is that a non-3n indel might be rescued via a frame-restoring splice form. Here we examine this hypothesis by inducing non-3n indels in many genes in rice and find that many non-3n indels are associated with a splice form that restores the reading frame. In the majority of these cases the indel appears to induce the potential rescuing splice form. We examine two top hit cases in detail and show functional rescue by splice modification. More generally, the frame-restoring forms are, however, low abundance and probably result in compromised proteins. We conclude then that splice mediated rescue is possible, but probably uncommon. Nonetheless it should not be overlooked in experimental design and interpretation.
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