ISM 2022 (Microscopy)


Asaf Ashkenazy-Titelman Mohammad Atrash Alon Boocholez Yaron Shav-Tal
The Mina & Everard Goodman Faculty of Life Sciences & Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel

The organization and compaction of mRNAs into an mRNP during nucleo-cytoplasmic transport and export, are key steps in gene expression. Changes in mRNA organization after release from the gene, during nucleoplasmic diffusion and in preparation for export, are unknown. It is assumed that at the nuclear pore complex (NPC), the mRNP unfolds and enters 5`-first into the pore. We revisit this issue to directly examine if this assumption holds as modus operandi in mammalian cells, using single molecule RNA FISH and advanced microscopy to detect the organization of the 5’, middle and 3’-ends of several long transcripts. We find that an mRNP is compact during nucleoplasmic travels compared to a more open structure after release from the gene. The mRNP is more open also in the nuclear periphery. Compaction levels of nuclear transcripts could be modulated by varying nuclear levels of SR proteins and by changing global genome structure. The nuclear mRNPs were mostly rod-shaped with distant 5` and 3`-ends, although for some, the transcript ends were in proximity suggesting a circular formation. The latter was much more abundant in the cytoplasm. Modifying the mRNA structure to distance the 5` and 3`-ends was only partly achieved by reducing the energetic status of the cell or inhibiting translation. Exit from the NPC was directly observed by labeling different parts of the same transcript, exhibiting predominant 5’-first export for both mRNAs and non-translating lncRNAs. Altogether, we show that the mRNP is a flexible structure during travels, with 5`-directionality during export.