Mechanisms regulating mRNA localization and localized translation

MRNA的细胞质运输和定位是一种转录后机制，它将蛋白质的合成限制在细胞内的特定位置。这一过程在多细胞生物体的发育、细胞的运动、细胞的不对称分裂和神经元的生长中起着重要的作用。我们使用萌芽酵母酿酒酵母作为模式生物，因为它具有简单和遗传上易驯化的优势，具有与后生动物相同的涉及mRNA定位和翻译控制的潜在机制。 在目前的芽期酵母基因定位模型中，芽定位的转录本首先在细胞核内被RNA结合蛋白She2识别，它结合了它们的定位元件(S)。翻译抑制物也被招募到核内的mRNA上。一旦进入细胞质，She2就与适配蛋白She3和V型肌球蛋白Myo4结合，形成一个复合体，沿着肌动蛋白细胞骨架向芽尖运输。一旦定位，磷酸化就释放了对信使核糖核酸的翻译抑制，允许它的局部翻译。 最近，对She2的磷酸化蛋白质组学分析发现，在She2的NLS中存在几个磷酸化残基，包括依赖于CK2的磷酸化位点。其中一些磷酸化事件抑制了She2的二聚化，支持了这些翻译后修饰的潜在调节作用。我们还发现，She2通过与RNA聚合酶II延伸因子Spt4-Spt5的相互作用，以共转录的方式被招募到芽定位的转录本中。目前尚不清楚She2如何与Spt4-Spt5相互作用，以及为什么需要这种相互作用。 我们假设She2和Spt4-Spt5之间的相互作用调节了She2与新生RNA邮政编码的亲和力。She2的磷酸化将调节其核进口和获得新生的RNA邮政编码。为了探索这些问题，我们提出了以下目标： 1)确定She2在转录过程中如何被招募到其靶mRNAs。我们将探索Spt4-Spt5和LOC1在新生转录本上招募She2中的作用。CHIP-SEQ实验将有助于确定SHE2和LOC1在全基因组范围内共转录招募的程度。 2)明确She2磷酸化在调节mRNA定位中的作用。我们将探索依赖于CK2的She2的磷酸化在调节其核输入和活性中的作用。我们将使用蛋白质组学来鉴定和表征作用于She2的其他激酶。 这个项目将揭示重要的机械学见解，即转录机制在参与mRNA定位的限制性商业惯例的共转录招募中所起的作用。此外，还将探索这些过程的新的调控水平，这将有助于理解细胞如何在空间和时间上控制其蛋白质的局部合成。

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.