Characterising (pre-)mrnp organisation at different stages of gene regulation using single-molecule microscopy
mRNAs act as the central molecules in gene regulation, helping convert the genetic information stored in the DNA to functional proteins. As a single-stranded polymer, hundreds to thousands of nucleotides in length, mRNAs can form extensive secondary and tertiary structures and, together with proteins, are packaged into assemblies called messenger ribonucleoproteins (mRNPs). The 3D organisation of (pre-)mRNPs influences many aspects of what happens to them, including regulating their processing, export and translation in the cytoplasm. Despite their significance, our understanding of the structural organisation of (pre-)mRNPs in vivo is minimal, as is our comprehension of the principles that govern it.  During my PhD, I have developed an RNA-centric view on (pre-)mRNP organisation. For this, I have established an approach combining single-molecule RNA in situ hybridisation (smFISH) with structured illumination microscopy (SIM) and used it to study mRNP organisation in the nucleus and cytoplasm. Our results suggest that (pre-)mRNP organisation is altered at various stages during its lifetime. We show that (pre-)mRNP packaging starts co-transcriptionally, with introns organised into compact conformations. This organisation is altered during the course of transcription as the polymerase travels along the gene, finally assembling an intron with the ends in proximity in a spliceosome dependent manner, suggesting that co-transcriptional intron organisation could be critical in determining its excision. Once released, mRNPs have a compact linear organisation in the nucleoplasm and possibly a rod-like conformation. mRNP organisation in the cytoplasm is influenced by its translational status. While translation opens up mRNPs, separating the ends of the mRNA, translation inhibition and release of ribosomes, or recruitment to stress granules result in mRNPs having a highly compact structure. Interestingly, we rarely find mRNAs with the 5’ and 3’ ends in proximity, suggesting that closed-looped translation is not a universal state for all translating mRNAs. Together, our results provide a unique and essential view of mRNP organisation in cells and reveal important insight into the role of mRNP conformation in regulating translation and mRNP processing.
mRNP pre-mRNP mRNP organization single molecule microscopy closed-loop model smFISH mRNP architecture mRNA structure 5’-3’ communication mRNA translation mRNA splicing RNA compaction RNA imaging super-resolution microscopy U1-Pol II tethering intron looping