Mechanisms regulating mRNA localization and localized translation

调节 mRNA 定位和本地化翻译的机制

• 批准号: RGPIN-2019-05023
• 负责人: Chartrand, Pascal
• 金额: $ 3.06万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738016
• 关键词: Mechanisms; regulating; mRNA; localization; localized

The cytoplasmic transport and localization of mRNA is a post-transcriptional mechanism that restricts the synthesis of proteins to specific sites within a cell. This process plays important roles in the development of multicellular organisms, in cell motility, in asymmetric cell division and in neuronal growth. We are using the budding yeast Saccharomyces cerevisiae, in which over thirty mRNAs are localized to the bud tip, as a model organism, as it offers the advantage of a simple and genetically tractable organism, which possesses the same underlying mechanisms involved in mRNA localization and translational control as in metazoans. In the current model of mRNA localization in budding yeast, bud-localized transcripts are first recognized in the nucleus by the RNA-binding protein (RBP) She2, which binds their localization element(s). Translational repressors are also recruited on the mRNA in the nucleus. Once in the cytoplasm, She2 binds the adaptor protein She3 and the type V myosin Myo4, forming a complex transported along the actin cytoskeleton toward the bud tip. Once localized, phosphorylation releases the translational repression on the mRNA, allowing its local translation. Recently, a phosphoproteomic analysis of She2 revealed the presence of several phosphorylated residues, including CK2-dependent phosphorylation sites in the NLS of She2. Some of these phosphorylation events inhibit the dimerization of She2, supporting a potential regulatory role for these post-translational modifications. We also found that She2 is recruited cotranscriptionally to bud-localized transcripts via its interaction with the RNA polymerase II elongation factor Spt4-Spt5. It remains unclear how She2 interacts with Spt4-Spt5 and why this interaction is required. We hypothesize that the interaction between She2 and Spt4-Spt5 modulates the affinity of She2 for binding nascent RNA zipcodes. Phosphorylation of She2 would regulate its nuclear import and access to nascent RNA zipcodes. To explore these questions, we propose the following objectives: 1) Determine how She2 is recruited to its target mRNAs during transcription. We will explore the roles of Spt4-Spt5 and Loc1 in the recruitment of She2 on nascent transcripts. ChIP-seq experiments will help determine the extent of She2 and Loc1 cotranscriptional recruitment genome-wide. 2) Identify the roles of She2 phosphorylation in the regulation of mRNA localization. We will explore the role of CK2-dependent phosphorylation of She2 in regulating its nuclear import and activity. We will use proteomics to identify and characterize other kinases acting on She2. This program will reveal important mechanistic insights on the role of the transcription machinery in the cotranscriptional recruitment of RBPs involved in mRNA localization. Furthermore, new levels of regulation of these processes will be explored, which will help understand how cells control, in space and time, the local synthesis of their proteins.

mRNA的胞质转运和定位是一种转录后机制，其将蛋白质的合成限制在细胞内的特定位点。这一过程在多细胞生物体的发育、细胞运动、不对称细胞分裂和神经元生长中起着重要作用。我们使用出芽酵母酿酒酵母作为模式生物，其中超过30种mRNA定位于芽尖，因为它提供了一种简单且遗传上易于处理的生物体的优势，其具有与后生动物中相同的参与mRNA定位和翻译控制的潜在机制。 在目前芽殖酵母中mRNA定位的模型中，芽定位的转录物首先在细胞核中被RNA结合蛋白（RBP）She 2识别，该蛋白结合其定位元件。翻译阻遏物也在细胞核中的mRNA上募集。一旦进入细胞质，She 2结合接头蛋白She 3和V型肌球蛋白Myo 4，形成复合物，沿着沿着肌动蛋白细胞骨架向芽尖运输。一旦定位，磷酸化释放mRNA上的翻译抑制，允许其局部翻译。 最近，一个磷酸化蛋白质组学分析的She 2揭示了存在几个磷酸化残基，包括CK 2依赖的磷酸化位点的NLS的She 2。这些磷酸化事件中的一些抑制She 2的二聚化，支持这些翻译后修饰的潜在调节作用。我们还发现，通过与RNA聚合酶II延伸因子Spt 4-Spt 5的相互作用，She 2被共转录地募集到芽定位的转录本。目前尚不清楚She 2如何与Spt 4-Spt 5相互作用以及为什么需要这种相互作用。 我们假设She 2和Spt 4-Spt 5之间的相互作用调节She 2结合新生RNA拉链的亲和力。She 2的磷酸化将调节其核输入和进入新生RNA拉链。为了探索这些问题，我们提出了以下目标：1）确定在转录过程中She 2是如何被募集到其靶mRNA的。我们将探讨Spt 4-Spt 5和Loc 1在新生转录本上招募She 2中的作用。ChIP-seq实验将有助于确定She 2和Loc 1在全基因组范围内共转录募集的程度。 2)确定She 2磷酸化在mRNA定位调节中的作用。我们将探讨CK 2依赖性磷酸化的She 2在调节其核输入和活性的作用。我们将使用蛋白质组学来鉴定和表征作用于She 2的其他激酶。 该计划将揭示重要的机制的见解的作用，转录机制的参与mRNA定位的RBP的共转录招聘。此外，将探索这些过程的新水平的调节，这将有助于了解细胞如何在空间和时间上控制其蛋白质的局部合成。