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

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.