Structural and Functional Studies of Ancient RNA-binding Sm Proteins

Randolph, Peter, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Mura, Cameron, Department of Chemistry, University of Virginia

The Sm superfamily’s central role in RNA processing and regulation, combined with their existence in all three domains, makes them a model system for exploring RNA processing evolution. Sm-mediated interaction between RNAs play vital roles in important pathways such as virulence, quorum sensing, cell death and aging, and mRNA splicing. The largest gap in our knowledge of the Sm superfamily is in the archaeal branch. Many archaeal systems can provide invaluable knowledge about their more complicated analogous eukaryal systems by supplying a simpler model to work with. Initial work on the Sm-like archaeal proteins (SmAPs) were crucial to our understanding of how Sm proteins oligomerize and bind RNA. Unfortunately, since this early work, the study of SmAPs has been limited, and SmAP in vivo functions are virtually unknown. Understanding these in vivo functions of SmAPs would allow us a better understanding of basic Sm protein function, provide a window into the evolution of the large eukaryal ribonucleoprotein complexes, and possibly link the evolution of bacterial Hfq and eukaryal Sm proteins.
The crenarchaea Pyrobaculum aerophilum is a deep-branching, hyperthermophile that encodes multiple SmAP paralogs. The two known Pae SmAP structures (SmAP1, SmAP3) illuminated Sm protein evolution and assembly, and implied that these homologs may represent an ancestral form of the complexes that developed into the extant heteromeric Sm assemblies of eukaryotes, such as those at the heart of the spliceosome. Our work on the final Pae SmAP, SmAP2, reveals that Pae SmAP2 oligomerizes as a unique octamer (unseen in previous SmAPs) in two rare space groups, and binds both A-rich and U-rich RNA reminiscent of the bacterial Hfq (chaperone). The crystal structure revealed that Pae SmAP2 lacks the conserved residues seen in the common U-rich and A-rich binding pockets of other Sm proteins, but does contain the aromatic (Tyr-42) necessary for lateral-rim binding. Further research is necessary to determine the specific binding mechanisms of Pae SmAP2···RNA binding, the Pae SmAP2 solution state, and determine the individual functions of the SmAP paralogs in Pyrobaculum aerophilum.
Many deep-branching bacteria share a high degree of similarity (genomically) with archaea, including the hyper-thermophilic Thermotoga maritima. T. maritima Hfq is an interesting homolog because of its simplicity (no C-terminal tail) and the aforementioned archaeal genome. The two studies reported here, one in archaea and one in bacteria, will help to illuminate the functions of ancient Sm proteins, supply a window into RNA processing in archaea, and the evolution of Sm proteins. In this study, we found that the putative Hfq homolog from the thermophillic Thermotoga maritima (Tma) heterologously co-purifies with U/C-rich nanoRNAs, binding with a nanomolar affinity. Identified nanoRNA sequences all contain a 5’ monophosphate and a 3’ hydroxyl and compete with U-rich sequences for the proximal face of Hfq. Data suggests that the position of cytosine within the sequence, rather than the absolute number of cytosines is the key factor in determining affinity. The crystal structure shows that, even under denaturing condition, a small amount of the heterologous nanoRNA remains bound. Tma Hfq forms a hexamer within the crystal, agreeing with previous studies on the functional form of Hfq in Escherichia coli (Eco) and other bacterial species. However, our studies of Tma Hfq suggest that an equilibrium exists between a homo-hexamer and a homo-dodecamer. Both oligomeric states are capable of binding poly-adenine and poly-uracil RNA with low nanomolar binding affinities, with poly-A and poly-U RNA preferentially binding to the dodecamer and hexamer, respectively. This leads to a shift in equilibrium between the states; poly-U shifting the equilibrium toward the hexameric state, and poly-A having no effect.

PHD (Doctor of Philosophy)
Sm proteins, RNA-binding, RNA, Hfq, Archaea, crystallography
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