Although the involvement of Ser/Arg-rich (SR) proteins in RNA metabolism is well documented, their role in vertebrate development remains elusive. We, therefore, elected to take advantage of the zebrafish model organism to study the SR genes' functions using the splicing morpholino (sMO) microinjection and the programmable site-specific nucleases. Consistent with previous research, we revealed discrepancies between the mutant and morphant phenotypes and we show that these inconsistencies may result from a large number of unsuspected inadvertent morpholino RNA targets. While microinjection of MOs directed against srsf5a (sMOsrsf5a) led to developmental defects, the corresponding homozygous mutants did not display any phenotypic traits. Furthermore, microinjection of sMOsrsf5a into srsf5a^−/− led to the previously observed morphant phenotype. Similar findings were observed for other SR genes. sMOsrsf5a alternative target genes were identified using deep mRNA sequencing. We uncovered that only 11 consecutive bases complementary to sMOsrsf5a are sufficient for binding and subsequent blocking of splice sites. In addition, we observed that sMOsrsf5a secondary targets can be reduced by increasing embryos growth temperature after microinjection. Our data contribute to the debate about MO specificity, efficacy and the number of unknown targeted sequences.

中文翻译：

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在丹尼奥里奥中吗啉代敲低的无意RNA靶标的数量被大大低估了：来自富含Ser / Arg的剪接因子研究的证据

尽管富含Ser / Arg的（SR）蛋白参与RNA代谢的文献已被充分证明，但它们在脊椎动物发育中的作用仍然难以捉摸。因此，我们选择利用斑马鱼模型生物利用剪接吗啉代（sMO）显微注射和可编程的位点特异性核酸酶研究SR基因的功能。与先前的研究一致，我们揭示了突变体和吗啡型表型之间的差异，并且我们表明这些不一致可能是由于大量意外的吗啉代RNA靶标引起的。虽然针对srsf5a（sMO srsf5a）的MO的显微注射导致发育缺陷，但相应的纯合突变体未显示任何表型性状。此外，sMO的显微注射srsf5a变成srsf5a ^-/-导致先前观察到的吗啡型表型。对于其他SR基因也观察到类似的发现。sMO srsf5a替代靶基因使用深层mRNA测序进行了鉴定。我们发现，只有11个与sMO srsf5a互补的连续碱基足以结合并随后阻断剪接位点。此外，我们观察到，sMO srsf5a的次要靶点可以通过显微注射后提高胚胎的生长温度来降低。我们的数据有助于就MO的特异性，功效和未知靶向序列的数量展开辩论。