CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs.

Tim Schneider,Oliver Rossbach,Stefan Starke,Stefan Reich,Lee-Hsueh Hung,Jan Medenbach,Silke Schreiner,Heinrich Eckhof,Albrecht Bindereif

doi:10.1038/srep31313

Tim Schneider, Oliver Rossbach + Show 7 more

Open Access

https://doi.org/10.1038/srep31313

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Journal: Scientific Reports	Publication Date: Aug 11, 2016
Citations: 131	License type: CC BY 4.0

Affiliation: University of Giessen, University of Regensburg

Abstract

Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component.

Highlights

Circular RNAs constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing
To identify circRNA-protein complexes, we subjected cytoplasmic extract (S100) from HeLa cells to glycerol gradient fractionation, whereby RNAs and RNPs sediment according to their molecular size and shape
We focussed the analysis on S100 extract, since circRNAs predominantly localize in the cytoplasm. 22 fractions were collected, followed by RNA preparation from every second fraction and RT-PCR assays for 12 relatively abundant circRNAs, based on circular-junction-specific primers

Summary

Introduction

Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. RNA-seq, and bioinformatic circular-junction analysis, to identify a subfamily of circRNAs stably associated with IMP3, followed by validation and further characterization of several examples of IMP3-containing circRNPs. This provides a general approach to identify circRNPs carrying a specific protein component.

Results

Conclusion