编辑推荐：
RNA 结合蛋白（RBPs）能与RNA分子相互作用，影响RNA分子稳定性，剪接，细胞核输出以及靶标转录序列的翻译。因此找到RBP-RNA是如何相互作用，对于了解基因表达，细胞功能等具有重要意义。
生物通报道：RNA
结合蛋白（RBPs）能与RNA分子相互作用，影响RNA分子稳定性，剪接，细胞核输出以及靶标转录序列的翻译。因此找到RBP-RNA是如何相互作用，对于了解基因表达，细胞功能等具有重要意义。
识别RBP靶标的一个常用房间就是核糖核苷交联和免疫共沉淀(Cross-linking and immunoprecipitation,
CLIP)，但是来自科罗拉多大学博尔德分校的Roy Parker
表示，一直以来都存在一个问题，那就是“如何能在小部分，特异性细胞亚型中完成这一实验，”毕竟微量样品很难进行生化实验。
Rosbash团队致力于研究生物钟的分子调控机制，特别是果蝇的时钟神经元中基因转录后修饰的调控机制。当他们利用生化方法分析生物钟调控相关的RNA结合蛋白以及相应的RNA结合谱时，发现现有方法需要大量的材料，而他们没有，而且他们想直接在特定亚群的神经元中标记与目标蛋白结合的RNA，并通过测序靶定。
为此他们研发出了一种称为Targets of RNA-binding proteins Identified By Editing
(TRIBE)的方法，这种方法能将脱氨酶ADAR的催化区域融合到目标蛋白上，通过识别被编辑的碱基来找到与蛋白相互作用的靶RNA。
研究组成功将这一技术应用到了三种RBPs上，并且显示能将范围缩小到150个果蝇神经元，不过Rosbash表示还能更少，“从理论上说，这完全能用于单细胞。”
不过研究人员还需要指导脱氨酶ADAR如何选择需要编辑的碱基位点。由于ADAR只能结合双链RNA，因此这种方法也会漏掉那些没有双链结构的靶RNA。
另外一个研究组：来自威斯康星-麦迪逊大学的Marvin Wickens 等人则设计了一个相似的技术：RNA
Tagging，这个技术能将RBPs与一种能在相关转录本末端添加尿苷的酶融合在一起。
研究组成员花费多年时间研究核酸转移酶，并意外地找到了一种能在转录本末端添加多聚尿苷U的RNA修饰酶。他们将这种多聚尿苷合成酶（PUP）融合在目标蛋白上，并且验证了其只在与目标蛋白结合的RNA的末端添加尿苷尾巴。这种RNA标签法并不需要特殊的RNA结构，只需要双端测序来识别出mRNA以及末端被添加的U的数目。
“这两种方法各有长处和短处，”Parker说，他建议可以将两者结合起来，同时观察两个RBP。
|
TAGGING TECHNIQUE |
RNA-BINDING PROTEIN FUSED WITH |
ENZYME FUNCTION |
SAMPLE PROCESSING FOR SEQUENCING |
SINGLE-CELL ANALYSIS |
|
Rna Tagging |
Poly(U) polymerase PUP-2 from C. elegans |
Adds uridines at the 3’ end of bound RNAs |
A specialized cDNA library is made with
“U-select” primers, which home in on U-tags. |
Theoretically possible, but because U-tag
library protocol is not a standard approach
for high-throughput sequencing, it may
need tweaking. |
|
TRIBE |
Catalytic domain of RNA-editing enzyme ADAR
from D. melanogaster |
Converts adenosines to inosines at sites
targeted
by RNA-binding proteins |
Standard RNA extraction
and preparation protocols
for RNA-seq |
Possible in principle; TRIBE uses the same
standard deep-sequencing technique that has
been applied to single cells. |
原文摘要：
TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of
RNA-Binding Proteins
RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current
methods for identifying in vivo targets of an RBP are imperfect and not amenable
to examining small numbers of cells. To address these issues, we developed TRIBE
(targets of RNA-binding proteins identified by editing), a technique that
couples an RBP to the catalytic domain of the Drosophila RNA-editing enzyme ADAR
and expresses the fusion protein in vivo. RBP targets are marked with novel RNA
editing events and identified by sequencing RNA. We have used TRIBE to identify
the targets of three RBPs (Hrp48, dFMR1, and NonA). TRIBE compares favorably to
other methods, including CLIP, and we have identified RBP targets from as little
as 150 specific fly neurons. TRIBE can be performed without an antibody and in
small numbers of specific cells.
