Structural Co-evolution of the LARP Superfamily and its Role in Functional Plasticity

项目总结/摘要 我们研究计划的长期目标是破译“非经典”蛋白质的分子机制- RNA相互作用研究了一个特别迷人的和疾病相关的蛋白质家族，称为La相关的 蛋白质（LARP）超家族，在分子水平上。细胞质LARP在转录后调控中起着关键作用， 通过调节mRNA的主动翻译、降解和储存之间的微妙平衡来进行基因控制。 因此，许多LARP与各种癌症和纤维增生性疾病密切相关，使得它们 一类重要的药物靶点然而，治疗的发展由于缺乏 详细的分子级信息。我们的研究将阐明RNA识别的分子机制 特别是，为了解释共性和个性的复杂性，即他们的共同点是如何表现出来的， 一个RNA结合基序，称为La-模块，已经单独进化为允许特异性RNA识别， 从而实现其独特的功能。这项调查是基于我们在溶液和固态方面的独特优势 NMR光谱及其与其他生物化学，生物物理，计算和功能的紧密耦合 接近。我们的初步努力将遵循两条调查路线，同时侧重于两名成员， LARP超家族，hLARP 6和hLARP 1。在研究的第一条线中，我们将探索hLARP 6的La-模块是如何 实现了高度保守的5'茎环（5' SL）基序的排他性识别，该基序存在于所有verte中， 编码I型胶原蛋白的哺乳动物mRNA。这项研究将提供一个详细的分子水平的地图， La-模块的单个元件有助于5 'SL结合的特异性和亲和力。详细的见解 将获得的数据，加上目前可用的生物化学和生物物理数据，将提供重要的见解 进入分子“共生”的个别元素的La模块和关闭这一关键差距的知识 这是开发针对纤维增生性疾病的治疗策略所需的。第二行中 在本研究中，我们将类似地剖析hLARP 1的La-模块如何识别一种明显不同类型的RNA 与hLARP 6相比。发现hLARP 1与增殖和细胞周期缺陷密切相关， 在恶性细胞和组织中显著上调。最近的生物化学研究表明，La- hLARP 1的模块依次与3 ′ poly（A）结合，然后与5 ′末端寡嘧啶（5 ′ TOP）基序结合 的mRNA。值得注意的是，这种特殊的两步行为尚未在任何其他LARP中观察到。我们将 通过研究hLARP 1的结构和动力学变化，揭示了这种不寻常的分子机制 在poly（A）和5 'TOP基序结合后。这项研究将首次揭示， 一个RNA与其La-模块的结合引发了第二个RNA靶标结合所需的结构和动力学变化。 总的来说，这两条线的比较研究将协同引导我们理解基本的 连接La-模块的单个元件的共同进化及其在RNA中的特定作用的原理 识别，从而解释了结构和动态可塑性如何有助于功能可塑性。

PROJECT SUMMARY/ABSTRACT The long-term goal of our research program is to decipher the molecular mechanism of “non-canonical” protein- RNA interactions studying a particularly fascinating and disease-relevant family of proteins, called the La-related protein (LARP) superfamily, on a molecular level. Cytoplasmic LARPs play a pivotal role in post-transcriptional gene control by regulating the delicate balance between active translation, degradation, and storage of mRNAs. Hence, many LARPs are intimately implicated in various cancers and fibroproliferative diseases rendering them an important class of druggable targets. However, the development of therapies has been stunted by the lack of detailed molecular-level information. Our research will elucidate the molecular mechanism of RNA recognition exhibited by LARPs, in particular, to explain the intricacies of commonality and individuality, i.e. how their com- mon RNA-binding motif, called the La-module, has individually evolved to allow specific RNA recognition and thus achieve its distinct function. The investigation is based on our unique strength in solution and solid-state NMR spectroscopy and their close coupling with other biochemical, biophysical, computational, and functional approaches. Our initial efforts will follow two lines of inquiry, simultaneously focusing on two members of the LARP superfamily, hLARP6 and hLARP1. In the first line of inquiry, we will explore how the La-module of hLARP6 achieves the exclusive recognition of the highly conserved 5' stem-loop (5'SL) motif, which is found in all verte- brate mRNAs encoding type I collagens. This line of study will provide a detailed molecular-level map on how individual elements of the La-module contribute to the specificity and affinity of 5'SL binding. The detailed insights to be gained, together with currently available biochemical and biophysical data, will provide essential insights into the molecular “symbiosis” of the individual elements of the La-module and close this critical gap in knowledge required for the development of therapeutic strategies against fibroproliferative diseases. In the second line of inquiry, we will analogously dissect how the La-module of hLARP1 recognizes a distinctly different type of RNA compared to hLARP6. hLARP1 was found to be heavily involved in proliferation and cell cycle defects and to be significantly upregulated in malignant cells and tissues. Very recent biochemical studies revealed that the La- module of hLARP1 sequentially binds to the 3' poly(A) and then to the 5ʹ terminal oligopyrimidine (5ʹTOP) motifs of mRNAs. Notably, this peculiar two-step behavior has not yet been observed for any other LARP. We will uncover this unusual molecular mechanism by investigating the structural and dynamic changes of hLARP1 upon binding of the poly(A) and 5'TOP motifs. This study will for the first time reveal how the initial binding of one RNA to its La-module elicits structural and dynamic changes required for the binding of a second RNA target. Overall, these two lines of comparative investigations will synergistically lead us to understand the fundamental principles that connect the co-evolution of the individual elements of the La-module and their specific role in RNA recognition, thus explaining how structural and dynamic plasticity contribute to functional plasticity.