Brugiolo Mattia, Botti Valentina, Liu Na, Müller-McNicoll Michaela, Neugebauer Karla M
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar St., New Haven, CT 06520, USA.
RNA Regulation Group, Cluster of Excellence 'Macromolecular Complexes', Goethe-University Frankfurt, Institute of Cell Biology and Neuroscience, Max-von-Laue-Str. 13, 60438 Frankfurt/Main, Germany.
Nucleic Acids Res. 2017 Oct 13;45(18):10452-10465. doi: 10.1093/nar/gkx671.
RNA binding proteins (RBPs) regulate the lives of all RNAs from transcription, processing, and function to decay. How RNA-protein interactions change over time and space to support these roles is poorly understood. Towards this end, we sought to determine how two SR proteins-SRSF3 and SRSF7, regulators of pre-mRNA splicing, nuclear export and translation-interact with RNA in different cellular compartments. To do so, we developed Fractionation iCLIP (Fr-iCLIP), in which chromatin, nucleoplasmic and cytoplasmic fractions are prepared from UV-crosslinked cells and then subjected to iCLIP. As expected, SRSF3 and SRSF7 targets were detected in all fractions, with intron, snoRNA and lncRNA interactions enriched in the nucleus. Cytoplasmically-bound mRNAs reflected distinct functional groupings, suggesting coordinated translation regulation. Surprisingly, hundreds of cytoplasmic intron targets were detected. These cytoplasmic introns were found to be highly conserved and introduced premature termination codons into coding regions. However, many intron-retained mRNAs were not substrates for nonsense-mediated decay (NMD), even though they were detected in polysomes. These findings suggest that intron-retained mRNAs in the cytoplasm have previously uncharacterized functions and/or escape surveillance. Hence, Fr-iCLIP detects the cellular location of RNA-protein interactions and provides insight into co-transcriptional, post-transcriptional and cytoplasmic RBP functions for coding and non-coding RNAs.
RNA结合蛋白（RBPs）从转录、加工、功能到降解调控着所有RNA的生命周期。目前对于RNA-蛋白质相互作用如何随时间和空间变化以支持这些功能还知之甚少。为此，我们试图确定两种SR蛋白——SRSF3和SRSF7（前体mRNA剪接、核输出和翻译的调节因子）如何在不同细胞区室中与RNA相互作用。为此，我们开发了分级分离iCLIP（Fr-iCLIP），即从经紫外线交联的细胞中制备染色质、核质和细胞质级分，然后进行iCLIP。正如预期的那样，在所有级分中都检测到了SRSF3和SRSF7的靶标，其中内含子、snoRNA和lncRNA相互作用在细胞核中富集。与细胞质结合的mRNA反映出不同的功能分组，表明存在协调的翻译调控。令人惊讶的是，检测到数百个细胞质内含子靶标。这些细胞质内含子被发现高度保守，并在编码区引入了提前终止密码子。然而，许多保留内含子的mRNA并非无义介导衰变（NMD）的底物，尽管它们在多核糖体中被检测到。这些发现表明，细胞质中保留内含子的mRNA具有以前未被表征的功能和/或逃避了监测。因此，Fr-iCLIP检测RNA-蛋白质相互作用的细胞定位，并为编码和非编码RNA的共转录、转录后和细胞质RBP功能提供了深入了解。
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