Spatial organization of transcribed genes in mammalian cells
哺乳动物细胞转录基因的空间组织
基本信息
- 批准号:422388934
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:德国
- 项目类别:Priority Programmes
- 财政年份:
- 资助国家:德国
- 起止时间:
- 项目状态:未结题
- 来源:
- 关键词:
项目摘要
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.
当前的资助提案是我之前的 SPP2202 研究项目的延续,该项目的重点是转录真核基因的空间组织。在过去的一段时间里,我们研究了几个长高表达的小鼠基因作为模型,并证明转录基因从其隐藏位点扩展并形成开放式转录环(TL),聚合酶沿着环移动并携带新生RNA。值得注意的是,TL 可以跨越微米,这与最近关于间期染色质是凝胶或固体的主张相悖。 TL 的延伸和形状表明了它们内在的刚性,我们将其归因于具有多个大量新生核糖核蛋白 (nRNP) 的高表达基因的密集修饰。刚度假设已通过实验和表达基因的聚合物建模成功得到检验。总之,我们的数据与流行的转录工厂模型相矛盾,并表明虽然微观上可解析的 TL 对于长的高表达基因是特异性的,但它们形成的机制可能代表真核转录的一般方面。该工作现已发表在《自然细胞生物学》杂志(2022)上。虽然之前的工作带来了关于表达基因的空间组织的明确信息,但它提出了有关真核转录的新的有趣问题,我将在新的资助期内解决这个问题。 (1) 关于剪接的规范观点是,它严格地共转录发生,内含子在读完后不久就被切除。我计划使用 Tg 基因作为模型,研究大规模转录情况下共转录剪接的速度。 (2) 之前对 TL 的观察是利用 FISH 在固定的组织或细胞中进行的。通过茎环/外壳蛋白系统靶向 Ttn 和 Cald1 基因的 nRNA,我计划对 TL 和转录爆发的时空动态进行活细胞观察。 (3) 由于 nRNP 和 mRNP 尺寸较小,因此对其结构知之甚少。根据我们初步的 EM 数据,我计划使用关联显微镜 (cryo-CLEM) 和冷冻电镜断层扫描来研究肌管中 Ttn 形成的 nRNP 和 TL 的 3D 结构。 (4)教科书上关于染色体区域是间期核的主要特征的知识并没有得到我们之前的研究的支持,这表明区域性可能仅仅是最后一次有丝分裂的结果。为了解决这个问题,我计划根据细胞中小鼠染色体的寡染色来评估领地性,这些染色体随着有丝分裂后时期的持续时间而变化。 (5) 最后,我计划完成 Tg 基因作为高强度转录上调模型的研究。特别是,我计划测试 Tg 昼夜节律性和 Tg 转录本中的内含子保留,作为分离甲状腺细胞中外分泌(甲状腺球蛋白分泌)和内分泌(激素产生)活动的可能机制。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Dr. Irina Solovei, Ph.D.其他文献
Dr. Irina Solovei, Ph.D.的其他文献
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{{ truncateString('Dr. Irina Solovei, Ph.D.', 18)}}的其他基金
Organization, ontogenetic differentiation and evolution of the inverted rod photoreceptor nuclei
倒杆光感受器核的组织、个体发育分化和进化
- 批准号:
165603048 - 财政年份:2010
- 资助金额:
-- - 项目类别:
Research Grants
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