Widespread specific intron-retention events in nuclear RNA complexes identified by sedimentation analysis of pluripotent cellular extracts

Many essential cellular processes require RNA to interact with protein(s) to form ribonucleic protein complexes (RNPs). For example, all cellular proteins are produced by the ribosome - a large and stable RNP, gene splicing requires a choreography of numerous small and large RNPs, even the replication of telomeric DNA requires an RNP. All these examples are stable RNPs that exhibit specific sedimentation rates (e.g., in a sucrose gradient) based on the composition of RNA and protein. In this study we aimed to identify RNAs components of RNPs on a transcriptome-wide scale. Using sucrose-gradient followed by sequencing we identified 1,057 RNA transcripts that are likely to be components of cellular RNPs. Our approach determined that RNP are not due to the cellular abundance nor length of the constituent RNA transcripts. The RNPs identified were predominantly nuclear, containing a specified retained intron and resistant to RNA degradation. Collectively our study reveals a widespread phenomena of a specific intron being retained in a stable nuclear RNP. Gradient ultracentrifugation was performed at 30,000 rpm (SW41 rotor) for 18h at 4°C. Sucrose gradients were fractionated using ISCO gradient fractionation system (ISCO Model 160 Gradient Former Foxy Jr. Fraction Collector) into 12 fractions of 900 ul. RNA was purified from sucrose fractions using Directzol RNA Miniprep kit (ZymoResearch, R2062), following the manufacturer’s instructions. Human IPSCs, various fractions.