Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve.
Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve.
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DOI：
10.1093/nar/gkv731
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发表时间：
2015-09-18
影响因子：
14.9
通讯作者：
Kramer S
中科院分区：
文献类型：
--
作者：
Fritz M;Vanselow J;Sauer N;Lamer S;Goos C;Siegel TN;Subota I;Schlosser A;Carrington M;Kramer S
RNP颗粒是核糖核蛋白组装体，在所有真核生物中调节mRNA的转录后命运。它们的确切功能仍然知之甚少，其中一个原因是RNP颗粒纯化尚未实现。我们已经利用锥虫的一个独特的功能，以制备一个细胞级分高度富集饥饿应激颗粒。首先，颗粒仍然被困在锥虫细胞骨架的笼状，膜下微管阵列，而可溶性蛋白质被冲走。第二，微管解聚并释放颗粒。RNA测序结合单分子mRNA FISH鉴定了编码核糖体mRNA的短且高度丰富的mRNA被排除在颗粒之外。通过质谱分析，我们已经确定了463个应激颗粒候选蛋白。对于通过eYFP标记测试的17/49个蛋白质，我们已经证实了定位于颗粒，包括一个磷酸酶，一个甲基转移酶和两个具有锥虫生命周期调节功能的蛋白质。这里提出的新方法能够无偏地识别新的RNP颗粒成分，为理解RNP颗粒功能铺平了道路。
RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.
克隆和表达Cruzi核糖体蛋白P0和抗P0自身抗体的表位分析。
影响因子：
15.3
作者：
SKEIKY, YAW;BENSON, DR;PARSONS, M;ELKON, KB;REED, SG
通讯作者：
REED, SG