EASTBIO Seeing double: how do dsRNA binding pseudoenzymes regulate vertebrate mRNAs?

前mRNAs和mRNAs在转录后受到包括剪接和编辑在内的各种不同机制的调控，这些机制改变了RNA的信息内容。锌指相关结构域又称DZF蛋白，是脊椎动物dsRNA结合蛋白家族中的四个成员：核因子90(NF90)、核因子45(NF45)、锌指RNA结合蛋白(ZFR)和精子细胞核周RNA结合蛋白(SPNR)。NF90、NF45和ZFR是哺乳动物出生后存活所必需的。我们已经证明了NF45与NF90、ZFR和SPNR以相同的保守界面在三个不同的复合体中相互作用。虽然它们的分子功能仍然难以捉摸，但ZFR和同系物是调节特定剪接事件的，而NF90可以通过反向剪接促进CircRNA的生物发生。此外，最近的数据表明，NF90和ZFR在通过作用于RNA的腺苷脱氨酶(ADAR)家族调节腺苷到肌苷的编辑方面具有相反的功能。我们希望解决的关键问题是：这些蛋白质是如何进化的，它们如何与高亲和力复合体中的dsRNA结合，它们如何改变剪接和编辑的结果？库克实验室先前的结构数据表明，DZF结构域是一个假酶结构域，在结构上与核苷酸转移酶如聚(A)聚合酶相似。NF90似乎通过结构域改组事件获得了dsRNA结合活性，其中两个dsRNA结合域是从ADAR2编辑酶获得的。与爱德华·华莱士的实验室合作，候选人将使用系统发生学方法重建这个蛋白质家族的进化史，以揭示酶活性丧失和结构域洗牌事件发生的时间。这种方法还将使我们能够提出假说，说明这些蛋白质是如何通过揭示获得的调节元件来调节的。我们还想了解NF90/NF45和ZFR/NF45复合体之间dsRNA结合特异性的差异。候选者将重建具有生理RNA靶标的蛋白质-RNA复合体，并结合各种结构方法(单粒子冷冻-EM、交联质谱、X射线结晶学)和其他生物物理方法来表征它们的结构和RNA结合活性。这些分析将得到系统发育研究和结构生物信息学分析的支持。总之，这些方法将使我们能够开发出关于这些蛋白质如何调节其RNA靶标的机械性假说。然后，这些假说将在体外和体内测试中得到验证。该项目将在充满活力的研究环境中提供系统发育分析、结构生物学和生物化学方法方面的优秀培训，重点放在基础细胞生物学上。

Pre-mRNAs and mRNAs are regulated post-transcriptionally by a variety of different mechanisms that include splicing and editing, which alter the information content of the RNAs. The "domain associated with zinc finger" or DZF proteins are a family of dsRNA binding proteins with four members in vertebrates: nuclear factor 90 (NF90), nuclear factor 45 (NF45), zinc finger RNA binding protein (ZFR) and spermatid perinuclear RNA binding protein (SPNR). NF90, NF45 and ZFR are each required for survival past birth in mammals. We have shown that NF45 interacts in three different complexes with NF90, ZFR and SPNR using the same conserved interface.While their molecular functions remain elusive, ZFR and homologues are regulate specific splicing events and NF90 can promote circRNA biogenesis by back-splicing. Furthermore, recent data suggest that NF90 and ZFR have opposing functions in regulating adenosine-to-inosine editing by a family of enzymes known as adenosine deaminases acting on RNA (ADARs). Key questions that we wish to address are: how did these proteins evolve, how do they bind to dsRNA in high affinity complexes, how do they alter outcomes of splicing and editing? Previous structural data from the Cook lab showed that the DZF domain is a pseudoenzyme domain, with structural similarities to nucleotidyltransferases such as poly(A) polymerase. NF90 appears to have acquired dsRNA binding activity through a domain shuffling event where two dsRNA binding domains were acquired from ADAR2 editing enzyme. In collaboration with Edward Wallace's lab, the candidate will use phylogenetic approaches to reconstruct the evolutionary history of this protein family to reveal when enzyme activity was lost and domain shuffling events occurred. This approach will also allow us to develop hypotheses about how these proteins are regulated by revealing acquired regulatory elements. We would also like to understand how dsRNA binding specificity differs between NF90/NF45 and ZFR/NF45 complexes. The candidate will reconstitute protein-RNA complexes with physiological RNA targets and integrate a variety of structural methods (single particle cryo-EM, cross-linking mass spectrometry, X-ray crystallography) and other biophysical methods to characterise their structures and RNA binding activity. These analyses will be supported by the phylogenetic studies and structural bioinformatic analysis. Together, these approaches will allow us to develop mechanistic hypotheses of how these proteins regulate their RNA targets. These hypotheses will then be tested used in vitro and in vivo assays. This project will provide excellent training in phylogenetic analysis, structural biology, and biochemistry methods in the context of a vibrant research environment with a strong focus on basic cell biology.