RNA processing is an essential cellular process that when dysregulated underlies the development of
neurological diseases. Several mutations in nuclease containing complexes cause pontocerebellar hypoplasia
(PCH), a severe neurological disorder that often leads to prenatal death. Most cases of PCH are linked to
mutations in the tRNA Splicing Endonuclease (TSEN) Complex, which is responsible for the cleavage of tRNA
introns prior to tRNA maturation, and its accessory protein, CLP1, which is a critical negative regulator of tRNA
splicing. Genetic deletion of any single TSEN protein in yeast, engineered to have tRNAs without introns, was
shown to be lethal, suggesting that the TSEN complex likely has substrates beyond the tRNAs, which may
underlie the development of PCH. Likewise, mutations in another nuclease, target of Egr 1 (TOE1), a
deadenylase and 3’-exonuclase, are also are linked to PCH. TOE1 is the only deadenylase believed to mature
snRNAs, but it also moonlights in other cellular pathways, highlighting how much is yet to understand about its
role in protein complexes.
To determine how mutations in TOE1, CLP1, and TSEN proteins lead to PCH, there remains a critical
need to understand how these complexes assemble, recognize and process RNAs, and how their enzymatic
activity is regulated. Characterizing healthy cellular roles of these proteins is essential to determining how their
dysfunction causes PCH. We aim to address these critical questions through the following proposed Aims. In
Aim 1, Structural and molecular techniques will be used to identify how the TSEN complex recognizes and
processes tRNAs and other RNA substrates. In Aim 2, we will determine how the CLP1/TSEN complex are
regulated at the molecular and cellular level and how PCH mutations disrupt their regulation. Further, in Aim 3,
we will identify how PCH mutations alter TOE1 function and regulation, using proteomics and molecular biology
The proposed work is significant because it will provide a structural description for how known PCH
mutations may interfere with complex formation, stability, or function for a range of PCH-linked proteins. This
work will further provide insight into shared mechanisms by which these protein complexes cause PCH.
Furthermore, the work here will characterize new RNA processing roles for these nucleases.