Spatial organization of transcribed genes in mammalian cells

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The current grant proposal is a continuation of my previous research project within SPP2202, which was focused on spatial organization of transcribed eukaryotic genes. In the course of the past period, we studied several long highly expressed mouse genes as models and demonstrated that a transcribed gene expands from its harboring locus and forms an open-ended transcription loop (TL) with polymerases moving along the loop and carrying nascent RNAs. Remarkably, TLs can span across microns arguing against recent propositions that interphase chromatin is a gel or a solid. Extension and shape of TLs suggest their intrinsic stiffness, which we attribute to dense decoration of highly expressed genes with multiple voluminous nascent ribonucleoproteins (nRNPs). The stiffness hypothesis was successfully tested experimentally and by polymer modeling of expressed genes. In summary, our data contradict the popular model of transcription factories and suggest that although microscopically resolvable TLs are specific for long highly expressed genes, the mechanisms underlying their formation could represent a general aspect of eukaryotic transcription. The work is now published in the journal Nature Cell Biology (2022). Whereas the previous work brought a clear message about spatial organization of expressed genes, it raised new intriguing questions concerning eukaryotic transcription, which I am going to address in the new funding period. (1) The canonical view on splicing is that it occurs strictly co-transcriptionally with introns being excised shortly after they are read through. Using the Tg gene as a model, I plan to study rapidity of co-transcriptional splicing in case of massive transcription. (2) The previous observations of TLs were performed utilizing FISH in fixed tissues or cells. By targeting nRNAs of the Ttn and Cald1 genes via the stem-loop/coat protein system, I plan live-cell observations of spatiotemporal dynamics of TLs and transcriptional bursting. (3) Very little is known about structure of nRNPs and mRNPs owing to their small size. Based on our preliminary EM data, I plan to study nRNPs and 3D structure of TLs formed by Ttn in myotubes, using correlative microscopy (cryo-CLEM) and cryo-EM tomography. (4) The textbook knowledge that chromosome territories are the major feature of an interphase nucleus is not supported by our previous works indicating that territoriality is likely a mere consequence of the last mitosis. To address this question, I plan to evaluate territoriality based on oligopainting of mouse chromosomes in cells that vary by duration of their postmitotic period. (5) And finally, I plan to complete the study of the Tg gene as a model for high and robust upregulation of transcription. In particular, I plan to test Tg circadian rhythmicity and intron retention in Tg transcripts as possible mechanisms for separation of exocrine (thyroglobulin secretion) and endocrine (hormone production) activities in thyrocytes.