Intron removal from pre-mRNAs is a critical step in the processing of RNA polymerase II transcripts, required to create translation competent mRNAs. In humans, introns account for large portions of the pre-mRNA, with intronic sequences representing about 95% of most pre-mRNA. Intron length varies considerably; introns can be as short as a few to hundreds of thousands of nucleotides in length. How nascent long intronic RNA is organized during transcription to facilitate the communication between 5′ and 3′ splice-sites required for spliceosome assembly however is still poorly understood. Here, we use single-molecule fluorescent RNA in situ hybridization (smFISH) to investigate the spatial organization of co- and post-transcriptional long introns in cells. Using two long introns within the POLA1 pre-mRNA as a model, we show that introns are packaged into compact assemblies, and when fully transcribed, are organized in a looped conformation with their ends in proximity. This organization is observed for nascent and nucleoplasmic pre-mRNAs and requires spliceosome assembly, as disruption of U2 snRNP binding results in introns with separated 5′ and 3′ ends. Moreover, interrogating the spatial organization of partially transcribed co-transcriptional POLA1 intron 35 indicates that the 5′ splice site is maintained proximal to the 3′ splice site during transcription, supporting a model that 5′ splice site tethering to the elongating polymerase might contribute to spliceosome assembly at long introns. Together, our study reveals details of intron and pre-mRNA organization in cells and provides a tool to investigate mechanisms of splicing for long introns.
Emerin modulates spatial organization of chromosome territories in cells on softer matrices
