An improved in vivo tethering assay with single molecule FISH reveals that a nematode Nanos enhances reporter expression and mRNA stability.
An improved in vivo tethering assay with single molecule FISH reveals that a nematode Nanos enhances reporter expression and mRNA stability.
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用单分子FISH进行的改进的体内拴系测定揭示了线虫Nanos增强了报告基因表达和mRNA稳定性。
DOI：
10.1261/rna.078693.121
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发表时间：
2021-06
期刊：
影响因子：
--
通讯作者：
Aoki ST
中科院分区：
文献类型：
--
作者：
Doenier J;Lynch TR;Kimble J;Aoki ST
稳健的方法对于测试 RNA 结合蛋白的体内调节机制至关重要。在这里，我们报告了蛋白质-mRNA 束缚测定的改进，以探测秀丽隐杆线虫成体种系中自然环境中 RNA 结合蛋白的功能。该测定依赖于从单个启动子表达两个 mRNA 的双报告基因，并通过反式剪接进行解析。 gfp 报告基因 3'UTR 含有 λN22 肽的功能性结合元件，而 mCherry 报告基因 3'UTR 携带突变的非功能元件。该策略能够通过免疫荧光对报告蛋白进行内部控制定量，并通过 smFISH 对 mRNA 进行内部控制定量。为了测试新系统，我们分析了线虫 Nanos 蛋白 NOS-3，它是生殖细胞命运的转录后调节因子。出乎意料的是，束缚的 NOS-3 增强了报告基因的表达。我们通过内源种系基因改造的第二个报告基因证实了这种增强活性。 NOS-3 报告基因表达的增强与其氨基末端固有无序区域相关，而不是与其羧基末端锌指相关。 RNA 定量显示，束缚的 NOS-3 增强了报告基因 mRNA 的稳定性。我们认为，这种直接的 NOS-3 增强活性可以解释一个悖论：传统上 Nanos 蛋白预计会抑制 RNA，但 nos-3 被发现可以促进 gld-1 表达，这种效应可能是直接的。无论如何，新的双报告基因显着改善了 RNA 结合蛋白束缚后报告基因表达的原位定量，以确定其在多细胞组织中的分子机制。
Robust methods are critical for testing the in vivo regulatory mechanism of RNA binding proteins. Here we report improvement of a protein–mRNA tethering assay to probe the function of an RNA binding protein in its natural context within the C. elegans adult germline. The assay relies on a dual reporter expressing two mRNAs from a single promoter and resolved by trans-splicing. The gfp reporter 3′UTR harbors functional binding elements for λN22 peptide, while the mCherry reporter 3′UTR carries mutated nonfunctional elements. This strategy enables internally controlled quantitation of reporter protein by immunofluorescence and mRNA by smFISH. To test the new system, we analyzed a C. elegans Nanos protein, NOS-3, which serves as a post-transcriptional regulator of germ cell fate. Unexpectedly, tethered NOS-3 enhanced reporter expression. We confirmed this enhancement activity with a second reporter engineered at an endogenous germline gene. NOS-3 enhancement of reporter expression was associated with its amino-terminal intrinsically disordered region, not its carboxy-terminal zinc fingers. RNA quantitation revealed that tethered NOS-3 enhances stability of the reporter mRNA. We suggest that this direct NOS-3 enhancement activity may explain a paradox: Classically Nanos proteins are expected to repress RNA, but nos-3 had been found to promote gld-1 expression, an effect that could be direct. Regardless, the new dual reporter dramatically improves in situ quantitation of reporter expression after RNA binding protein tethering to determine its molecular mechanism in a multicellular tissue.
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影响因子：
4.5
作者：
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