Deciphering the role of nuclear organization in mRNA and lncRNA nuclear retention

我们研究计划的长期目标是开发和应用定量，单分子和超分辨率显微镜RNA和蛋白质成像方法，短期目标是研究核组织在调节mRNA和长非编码RNA（lncRNA）的选择性核保留中的作用。在真核细胞中，通过将染色体包装在一个单独的细胞隔室（细胞核）中，遗传信息存储的位点与蛋白质合成的位点分开。这种分离需要一种复杂机制的共同进化，这种机制介导两个隔室之间蛋白质和RNA的交换。在这种调控框架内，mRNA作为信使分子，在细胞核中合成后，被转运到细胞质进行蛋白质翻译。除了mRNA之外，真核生物基因组被转录成具有很少或没有编码潜力的各种各样的RNA，其中最大且可能最多样化的组通常被概括为lncRNA。大多数lncRNA与mRNAs共有许多特征，然而，尽管它们在加工和组成方面与mRNAs相似，但大多数lncRNA保留在细胞核中。此外，最近的研究表明，在某些条件下，mRNA也可以保留在核中，这表明核保留可能是一个重要的调节机制，调节基因表达的不同方面;然而，调节lncRNA和mRNA的选择性保留的机制目前还不清楚。在各种核隔室内的隔离被认为在保留中起作用，然而，这个过程没有得到很好的研究，并且在机制上定义不清。因此，我们的目标是开发和应用定量活细胞和单分子显微镜方法和测定，这将使我们能够研究介导核RNA保留的机制，并研究不同核隔室在这一过程中的作用。我们推测，不同的核隔室通过不同的机制调节保留。虽然核斑点（NP）中的保留可能是由RNA与NP组分的频繁动态相互作用介导的瞬时过程，允许分子在NP内和NP之间扩散，但旁斑点中的保留可能通过由RNA支架促进的静态保留来实现，从而能够在单个旁斑点内长期隔离。完成这个项目将揭示RNA保留的基本机制和不同隔室在这一过程中的作用-迄今为止尚未得到重视和研究不足的基因调控方面-从而更好地理解基因表达的基础生物学。它还将导致创建研究细胞中RNA的定量工具，这些工具可用于研究RNA代谢的各个方面。

The long-term objective of our research program is to develop and apply quantitative, single molecule and super-resolution microscopy RNA and protein imaging approaches, with the short-term objective to study the role of nuclear organization in modulating the selective nuclear retention of mRNAs and long-noncoding RNAs (lncRNAs). In eukaryotic cells, the site of genetic information storage is separated from the site of protein synthesis by packaging chromosomes in a separate cellular compartment, the nucleus. This separation required the co-evolution of a complex machinery that mediates the exchange of proteins and RNA between the two compartments. Within this regulatory framework, mRNA serves as a messenger molecule that, after synthesis in the nucleus, is transported to the cytoplasm for protein translation. In addition to mRNAs, eukaryotic genomes are transcribed into a vast variety of RNAs with little or no coding potential, the largest and possibly most diverse group of which is often summarized as lncRNAs. Most lncRNAs share many features with mRNAs, however, despite their similarities to mRNAs in terms of processing and composition, most lncRNAs are retained in the nucleus. Moreover, recent studies indicate that mRNAs can also be nuclear retained under certain conditions, suggesting that nuclear retention might be an important regulatory mechanism that modulates different aspects of gene expression; however, the mechanisms that modulate selective retention of lncRNAs and mRNAs are currently not understood. Sequestration within various nuclear compartments is thought to play a role in retention, yet, this process is not well studied and mechanistically ill defined. Our objective is therefore to develop and apply quantitative live-cell and single molecule microscopy approaches and assays that will allow us to study the mechanisms that mediate nuclear RNA retention and investigate the role of different nuclear compartments in this process. We hypothesize that different nuclear compartments modulate retention through different mechanisms. While retention in nuclear speckles (NP) may be a transient process that is mediated by frequent dynamic interactions of RNAs with NP components, allowing diffusion of molecules within and between NPs, retention in paraspeckles might be achieved by static retention facilitated by an RNA scaffold, enabling long term sequestration within a single paraspeckle. Completing this project will shed light onto the fundamental mechanisms of RNA retention and the role of different compartments in this process - a so far unappreciated and poorly studied aspect of gene regulation - leading to a better understanding of the underlying basic biology of gene expression. It will furthermore lead to the creation of quantitative tools to study RNA in cells that can be applied to investigate various aspects of RNA metabolism.