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RNA和蛋白质成像方法，短期目标是研究核组织在调节mRNAs和长非编码RNA(LncRNAs)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室，即细胞核，将遗传信息存储的位置与蛋白质合成的位置分开。这种分离需要一种复杂的机制的共同进化，该机制调节两个隔室之间的蛋白质和RNA交换。在这个调控框架内，mRNA作为信使分子，在细胞核中合成后，被运输到细胞质进行蛋白质翻译。除了mRNAs外，真核生物基因组还转录成大量几乎没有编码潜力的RNAs，其中最大也可能是最多样化的一组通常被概括为lncRNAs。然而，大多数lncRNAs与mRNAs有许多相似的特征，尽管它们在加工和组成方面与mRNAs相似，但大多数lncRNAs保留在细胞核中。此外，最近的研究表明，在某些条件下，mRNAs也可以被核保留，这表明核保留可能是调节基因表达不同方面的一种重要调控机制；然而，调控lncRNAs和mRNAs选择性保留的机制目前尚不清楚。在不同的核隔室中的隔离被认为在保持中发挥作用，然而，这一过程没有得到很好的研究，机械上也没有明确的定义。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分析，使我们能够研究介导核RNA保留的机制，并研究不同的核隔室在这一过程中的作用。我们假设不同的核区通过不同的机制调节保持。虽然在核斑点(NP)中的滞留可能是一个短暂的过程，它是由RNA与NP组分频繁的动态相互作用介导的，允许分子在NP内部和之间扩散，但在类核斑中的滞留可能是通过RNA支架促进的静态滞留实现的，从而能够在单个核斑点内实现长期隔离。完成这个项目将阐明RNA保留的基本机制，以及不同间隔在这一过程中的作用--这是基因调控中迄今未被认识且研究较少的一个方面--有助于更好地理解基因表达的基本生物学基础。它还将导致创建定量工具来研究细胞中的RNA，这些工具可以应用于研究RNA新陈代谢的各个方面。

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.