Function of the Ubiquitin-Like Protein Hub1

Hub1是一种高度保守的泛素样蛋白，仅由泛素折叠组成。与典型的泛素样修饰剂不同，Hub1仅与蛋白质非共价相互作用。遗传学研究表明Hub1在RNA剪接中的作用，但其确切功能仍不明确。我们发现Hub1与一个称为HIND的元件结合，该元件存在于某些剪接体蛋白（Snu66或Prp38）中。Hub1-HIND复合物的x射线结构揭示了一种新的相互作用模式。Hub1的结合改变了剪接体，但几乎不影响一般剪接。然而，hub1修饰的剪接体获得了识别非规范5 ' -剪接位点的能力。在酵母中，SRC1通过两个非规范的5 '剪接位点进行选择性剪接，这需要hub1修饰的剪接体。有趣的是，无论Hub1是与Snu66或Prp38上的HIND元件结合，还是与这些剪接蛋白线性融合，它都能发挥作用。因此，Hub1不调节其直接靶标，而是一般地影响剪接体。我们计划进行遗传筛选，以确定对hub1依赖性剪接位点选择产生积极或消极影响的成分。由于Hub1， Snu66和HINDs是高度保守的，我们也会问Hub1的功能是否保守。我们确定了Hub1上的残基，这些残基对剪接或细胞活力至关重要，因此我们将寻找关键的结合伙伴。除了对Hub1功能的完整描述外，我们期望所提出的研究还将对泛素样蛋白的机制以及它们如何影响蛋白质活性提供一般的见解。

Hub1 is a highly conserved ubiquitin-like protein, which consists solely of the ubiquitin fold. Unlike canonical ubiquitin-like modifiers, Hub1 interacts with proteins only non-covalently. Genetic studies have suggested a role of Hub1 in RNA splicing, but its precise function remained poorly defined. We found that Hub1 binds to an element termed HIND, which is present in certain spliceosomal proteins (Snu66 or Prp38). The X-ray structure of a Hub1-HIND complex revealed a novel mode of interaction. Binding of Hub1 alters the spliceosome but barely affects general splicing. However, Hub1-modified spliceosomes gain the ability to recognize non-canonical 5’-splice sites. In yeast, SRC1 is alternatively spliced via two non-canonical 5’-splice sites, and this requires Hub1-modified spliceosomes. Interestingly, Hub1 functions regardless whether it binds to an HIND element on Snu66 or Prp38, or even when fused linearly to these splicing proteins. Thus Hub1 does not modulate its direct target, but rather affects the spliceosome in general. We plan to conduct genetic screens to identify components that affect Hub1-dependent splicesite selection positively or negatively. As Hub1, Snu66 and HINDs are highly conserved, we will also ask whether Hub1’s function is conserved. We identified residues on Hub1 that are crucial for splicing or for its essential role for cell viability, and thus we will search for the crucial binding partners. In addition to a full description of the function of Hub1, we expect that the proposed research will also provide general insights into the mechanisms of ubiquitin-like proteins and how they influence protein activities.