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Nucleic Acids Res. 2017 Oct 13;45(18):10452-10465. doi: 10.1093/nar/gkx671.

Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm.

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar St., New Haven, CT 06520, USA.
2
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.

Abstract

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.

PMID:
28977534
PMCID:
PMC5737842
DOI:
10.1093/nar/gkx671
[Indexed for MEDLINE]
Free PMC Article

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