mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.
mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.
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DOI：
10.1371/journal.pgen.1007105
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发表时间：
2017-11
期刊：
影响因子：
4.5
通讯作者：
Farber SA
中科院分区：
文献类型：
--
作者：
Anderson JL;Mulligan TS;Shen MC;Wang H;Scahill CM;Tan FJ;Du SJ;Busch-Nentwich EM;Farber SA
随着基于模式生物的研究从正向遗传学方法转向反向遗传学方法，主要是由于基因组编辑技术的简便性，在预测会对编码蛋白产生有害影响的突变中观察到异常表型的频率较低。在斑马鱼中，这种低频率的部分原因是由冗余或类似功能的基因的补偿，通常是由于脊椎动物中的硬骨鱼特异性全基因组复制的额外一轮。在这里，我们提供了额外的解释突变表型的频率低。我们分析了7个斑马鱼品系的mRNA加工，这些斑马鱼品系具有通过CRISPR/Cas9或ENU诱变方法产生的预期破坏基因功能的突变。7个细胞系中有5个显示出mRNA加工改变的证据：1个通过跳过的外显子不会导致移码，1个通过无义相关剪接不会导致移码，3个通过使用隐蔽剪接位点。这些结果强调了在做出结论或开始假设由于给定的基因组变化而丧失功能的研究之前，需要对突变系中产生的mRNA进行系统分析。此外，识别可能发生的适应类型可能会为突变体产生的策略提供信息。最近兴起的反向遗传，基因靶向方法使研究人员能够容易地在任何感兴趣的基因中产生相对容易的突变。如果这些突变对mRNA和编码的蛋白质具有预测的影响，我们将预期比通常在反向遗传筛选中看到的更多的异常表型。在这里，我们通过研究斑马鱼的七个独立突变来探索这种差异的一些原因。我们提出的证据表明，彻底的cDNA序列分析是一个关键步骤，在评估的可能性，一个给定的突变将产生亚纯型或无效等位基因。这项研究表明，突变背景下的mRNA加工通常会产生逃避无义介导的衰变的转录本，从而可能保留基因功能。通过了解细胞避免突变有害后果的方式，研究人员可以更好地设计反向遗传策略，以增加基因破坏的可能性。
As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation. The recent rise of reverse genetic, gene targeting methods has allowed researchers to readily generate mutations in any gene of interest with relative ease. Should these mutations have the predicted effect on the mRNA and encoded protein, we would expect many more abnormal phenotypes than are typically being seen in reverse genetic screens. Here we set out to explore some of the reasons for this discrepancy by studying seven separate mutations in zebrafish. We present evidence that thorough cDNA sequence analysis is a key step in assessing the likelihood that a given mutation will produce hypomorphic or null alleles. This study reveals that mRNA processing in the mutant background often produces transcripts that escape nonsense-mediated decay, thereby potentially preserving gene function. By understanding the ways that cells avoid the deleterious consequences of mutations, researchers can better design reverse genetic strategies to increase the likelihood of gene disruption.
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影响因子：
14.9
作者：
Cartegni, L;Wang, JH;Krainer, AR
通讯作者：
Krainer, AR
影响因子：
14.9
作者：
Kapustin Y;Chan E;Sarkar R;Wong F;Vorechovsky I;Winston RM;Tatusova T;Dibb NJ
通讯作者：
Dibb NJ
影响因子：
7
作者：
Jagannathan S;Bradley RK
通讯作者：
Bradley RK
影响因子：
3.7
作者：
Lalonde S;Stone OA;Lessard S;Lavertu A;Desjardins J;Beaudoin M;Rivas M;Stainier DYR;Lettre G
通讯作者：
Lettre G
DOI：
10.1242/dev.115584
发表时间：
2014-12
期刊：
Development (Cambridge, England)
影响因子：
--
作者：
Irion U;Krauss J;Nüsslein-Volhard C
通讯作者：
Nüsslein-Volhard C