Research Projects

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RNA Dynamics

As a colossal machine required for gene expression and comprising both RNA and protein parts, the spliceosome shares many similarities with the ribosome. In particular, both utilize RNA parts for substrate recognition and catalysis. Further, both require elaborate assembly pathways dependent on numerous DExD/H box proteins that belong to a ubiquitous super family of ATPases required in essentially all RNA-dependent processes to remodel RNA-RNA and RNA-protein structure.

However, whereas the ribosome assembles once and then functions over and over again, the spliceosome must assemble anew on each and every splicing substrate and later disassemble once splicing is complete. As a consequence, spliceosome assembly is intimately coupled to substrate recognition.  Further, the spliceosome must rearrange  the  substrate  in  between the chemical steps of splicing and release the splicing products. Eight DExD/H box ATPases underlie much of these dynamics and must act at specific stages to promote  splicing  in  a  productive  manner.   

We  have focused on determining the mechanisms for regulating these ATPases, defining the snRNA dynamics that these ATPases promote, and, as a consequence, inferring the structure and function of the snRNAs at precise stages in splicing. We have found, for example, that a GTPase and ubiquitin cooperate to regulate the ATPase Brr2, which catalyzes the most dramatic RNA rearrangement in splicing – the release of U4 snRNA from the catalytic snRNA U6 (Small et al., 2006, Mol Cell; Bellare et al., 2008, NSMB; Nielsen & Staley, 2012, G&D). 

Importantly, a number of splicing factors are mutated in a common form of blindness, retinitis pigmentosa (RP), and we have found in collaborative work that the human Brr2 gene is also mutated in RP and that these mutations compromise release of U4 snRNA, identifying this key step in spliceosome assembly as sensitive to disease mutations (Zhao et al., 2009, Am J Hum Genet).

By investigating the final ATPase required in spliceosome assembly, we have found evidence that the catalytic conformation of the spliceosome is already formed at this stage, implying that the final stage is dedicated to substrate positioning (Wlodaver & Staley, 2014, RNA).

Finally, by investigating the DExD/H box ATPase required to rearrange the substrate in between the chemical steps, we found a surprising requirement for a toggling rearrangement in U2 snRNA that couples to transient dismantling of the catalytic core, two rearrangements that likely promote substrate repositioning (Hilliker et al, 2007, G&D; Mefford & Staley, 2009, RNA). Still, many mysteries remain to be solved before we can boast a deep understanding of snRNA function, snRNA dynamics, and the role of DExD/H box ATPases in these dynamics