SPIDR:一种用于绘制RNA-蛋白相互作用的高度多重化方法,揭示了在细胞应激时选择性翻译抑制的潜在机制
Erica Wolin1, Jimmy K Guo2,3, Mario R Blanco2
1Department of Biological Sciences, Columbia University, New York City, New York 10027, USA.
bioRxiv : the preprint server for biology
|June 19, 2023
概括
我们开发了SPIDR,这是一种新的方法,可以在规模上绘制与RNA的RNA结合蛋白 (RBP) 相互作用的地图. SPIDR同时对数百个RBP-RNA位进行分析,揭示了mTOR抑制时的动态结合变化.
科学领域:
- 分子生物学分子生物学
- 基因组学就是基因组学.
- 生物化学 生物化学
背景情况:
- RNA结合蛋白 (RBPs) 调节mRNA处理和非编码RNA功能.
- 识别特定的RBP-RNA相互作用至关重要,但目前的方法具有挑战性.
- 像CLIP-seq这样的现有技术仅限于一次分析一个RBP.
研究的目的:
- 开发一种高通量方法,同时分析多个RBP-RNA相互作用.
- 为了克服当前CLIP-seq方法中单个RBP分析的局限性.
- 为了使RNA-蛋白相互作用的大规模发现.
主要方法:
- 开发了SPIDR (RBP目标的分割和聚合识别),一种大规模多重复合技术.
- 使用分池条形码和抗体-珠条形码来提高吞吐量.
- 与传统的CLIP方法相比,实现了两级的吞吐量增加.
主要成果:
- SPIDR同时识别了多个RBP的精确的单核酸RNA结合点.
- 在mTOR抑制过程中探索RBP结合变化.
- 发现4EBP1在mTOR抑制时动态结合抑制的mRNA的5'-未翻译区域.
结论:
- 通过SPIDR,可以同时分析数百个RBP-RNA相互作用,从而彻底改变了RNA生物学研究.
- 确定了一种用于mTOR介导的翻译控制的新机制,涉及动态4EBP1结合.
- SPIDR促进了RNA-蛋白相互作用的快速,大规模发现,以了解基因调节.
相关概念视频
01:29Translational Regulation
45
Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
45
01:53RNA Stability
33.7K
Intact DNA strands can be found in fossils, while scientists sometimes struggle to keep RNA intact under laboratory conditions. The structural variations between RNA and DNA underlie the differences in their stability and longevity. Because DNA is double-stranded, it is inherently more stable. The single-stranded structure of RNA is less stable but also more flexible and can form weak internal bonds. Additionally, most RNAs in the cell are relatively short, while DNA can be up to 250 million...
33.7K
01:23Types of RNA
64.1K
Overview
Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use.
RNA...
Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use.
RNA...
64.1K
01:23Regulation of Expression at Multiple Steps
951
The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. Transcriptional regulation is mediated by regulatory proteins such as transcription factors, activators, or repressors—these control gene expression by initiating or inhibiting the transcription of genes. Once a precursor or pre-mRNA is produced, it undergoes post-transcriptional modification, including 5' capping, splicing, and the...
951
02:51mRNA Stability and Gene Expression
5.7K
The structure and stability of mRNA molecules regulates gene expression, as mRNAs are a key step in the pathway from gene to protein. In eukaryotes, the half-life of mRNA varies from a few minutes up to several days. mRNA stability is essential in growth and development. The absence of the proteins regulating its stability, such as tristetraprolin in mice, can cause systemic issues, including bone marrow overgrowth, inflammation, and autoimmunity.
Cis-acting Elements involved in mRNA stability
Cis-acting Elements involved in mRNA stability
5.7K
02:31Nuclear Export of mRNA
7.7K
Before mRNAs are exported to the cytoplasm, it is crucial to check each mRNA for structural and functional integrity. Eukaryotic cells use several different mechanisms, collectively known as mRNA surveillance, to look for irregularities in mRNAs. Irregular or aberrant mRNA are rapidly degraded by various enzymes. If a defective mRNA escapes the surveillance, it would be translated into a protein which would either be non-functional or not function properly. One of the primary irregularities in...
7.7K