Functional Interrogation of Brain Microproteins

人类大脑必须利用非凡的蛋白质组多样性来接收、处理和执行复杂的认知任务。迄今为止，虽然许多研究都集中在人类大脑发育中的基因组和转录组调控上，但我们对这方面的蛋白质翻译的理解是有限的。因此，我的研究计划的长期目标是了解mRNA翻译在神经发育过程中是如何被调节的，以及蛋白质进化如何促进人类大脑的复杂性。最近的研究表明，蛋白质组的多样性远远超过之前的认识，部分原因是编码微蛋白=100个氨基酸的小开放阅读框（sorf）的广泛活跃翻译。在其他组织中，研究人员已将一系列功能归因于一小部分微蛋白，包括作为细胞内和细胞外第二信使、酶的变构调节因子和大分子复合物的组分。脑编码微蛋白的功能和生理意义尚不清楚，但它们代表了神经科学领域一个令人兴奋的、尚未被描述的前沿。为了鉴定人脑中的候选微蛋白，我们利用核糖体分析绘制了人类皮层从产前到成年生活的翻译景观。此外，我们同样绘制了人类胚胎干细胞（hESC）衍生神经元的“翻译组”。根据这些数据，我们确定了核糖体跨密码子的运动，并鉴定了蛋白质编码orf。我们的数据揭示了数千个编码微蛋白的sorf从注释为非编码的转录本中翻译出来。我们发现，大多数微蛋白是人类新进化出来的，其中许多是大脑特异性的。有趣的是，我们发现数百种微蛋白在功能上与内在无序RNA结合蛋白的RGG结构域相关，这表明这些微蛋白可能结合RNA来调节RNA的代谢或功能。这强调了识别脑表达微蛋白的蛋白质编码潜力的重要性，这需要在单个候选基因水平上进一步研究。我们假设嵌入基因组“非编码”区域的微蛋白子集对神经元的存活和功能至关重要。为了验证这一假设，我们提出以下具体目标：表征候选微蛋白在人类神经元中的定位和相互作用，目的是阐明生物功能。2.在人类神经元中进行CRISPR筛选，以鉴定在注释的非编码区域编码的高可信度功能微蛋白。总之，这些实验将确定神经元功能和存活的新调节因子，更重要的是，为神经系统中以前未被重视的一类分子提供新的见解。

The human brain must leverage extraordinary proteomic diversity to receive, process, and execute complex cognitive tasks. To date, while many studies have focused on genomic and transcriptomic regulation in the developing human brain, our understanding of protein translation in this context is limited. Therefore, the long term goal of my research program is to understand how mRNA translation is regulated during neurodevelopment, and how protein evolution contributes to human brain complexity. Recent studies suggest that the proteome is far more diverse than previously appreciated, due in part to the widespread active translation of small open reading frames (sORFs) encoding microproteins =100 amino acids. A range of functions have been ascribed to the small set of microproteins that have been studied in other tissues, including roles as intra- and extracellular second messengers, allosteric regulators of enzymes, and components of macromolecular complexes. The function and physiologic significance of brain-encoded microproteins is unknown, but they represent an exciting and as-yet uncharacterized frontier in neuroscience. To identify candidate microproteins in the human brain, we mapped the translational landscape of the human cortex, from prenatal to adult life, using ribosome profiling. Moreover, we have similarly mapped the `translatome' of human embryonic stem cell (hESC)-derived neurons. From this data, we determined the movement of ribosomes across codons and identified protein-coding ORFs. Our data has revealed the translation of thousands of sORFs, which encode microproteins, from transcripts annotated as non-coding. We found that the majority of microproteins are newly evolved in humans, and many are brain-specific. Interestingly, we found hundreds of microproteins that are functionally related to the RGG domain of intrinsically disordered RNA-binding proteins, suggesting that these microproteins may bind RNA to regulate RNA metabolism or function. This underscores the importance of discerning the protein coding potential of brain-expressed microproteins, which requires further investigation at the level of individual gene candidates. We hypothesize that a subset of microproteins embedded in `non-coding' regions of the genome are critical to neuron survival and function. To test this hypothesis, we propose the following Specific Aims: 1.Characterize the localization and interactome of candidate microproteins in human neurons, with the goal of elucidating biologic function. 2.Perform a CRISPR screen in human neurons to identify high-confidence functional microproteins encoded within annotated non-coding regions. Altogether, these experiments will identify novel regulators of neuron function and survival, and more importantly, provide new insight into a previously under-appreciated class of molecules in the nervous system.