Salazar, Anna Maria (2008) A Pumilio domain that forms heritable amyloid aggregates in yeast can regulate Pumillio-mediated translational repression in Drosophila. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-10012007-011631
Numerous human diseases have been described in which defects in protein folding pathways play a role in the development of a disease state. A subset of these diseases result in a decrease in the level of the active, native conformation of the protein. This could arise from several mechanisms, including an increase in degradation caused by misfolding, alterations in trafficking of the misfolded protein, or aggregation of the protein with a consequent decrease in the soluble, active form of the protein. At least 40 human diseases that fit into this last category, and are associated with the formation of amyloid fibers, deposits, or inclusions, have been characterized. Domains rich in glutamine (Q) and asparagine (N) are one class of sequences that seem to possess an affinity to form amyloids under native conditions. These domains are present in many metazoan proteins, including 472 in Drosophila and 143 in C. elegans. Q/N domains are found in several yeast prions. It is hypothesized that these domains may have been positively selected during evolution, perhaps in order to allow reversible switching of the functional domain of the protein into an inactive aggregated state. We wondered if this type of selection might also maintain Q/N domains in metazoans. The Drosophila melanogaster and Caenorhabditis elegans proteomes were searched for predicted proteins that contain nucleic acid binding domains (for RNA, DNA or both), and Q/N-rich sequences, using a threshold of 30 Q/Ns in 80 residues. One of the two strong Drosophila candidates is the translational repressor Pumilio (Pum). Earlier work by our group (Menon et al., Neuron 44, 663-676 (2004)) had shown that Pum is localized to the postsynaptic side of the larval neuromuscular junction (NMJ), where it acts as a regulator of local mRNA translation. In pum mutants, synaptic morphology is altered and GluRIIa is dramatically upregulated. This study shows that a Q/N-rich domain (denoted NQ1) from Pum is able to form ordered aggregates in budding yeast and is able to recapitulate the activity of a yeast prion, New1p: including visible aggregates, heritable phenotypic switching, and reversibility of an induced yeast prion state, [Psi+], by guanidine hydrochloride. To test whether NQ1 aggregate formation can perturb Pum's function in the nervous system, transgenic fly lines in which NQ1 expression is driven by GAL4 were created. Postsynaptic NQ1 expression generates alterations in the NMJ that phenocopy the pum loss-of-function phenotype and interact genetically with pum mutations. This is observed through an increase in type 1s and type 1b boutons and an increase in GluRIIa at the NMJ. Postsynaptic Pum overexpression in muscles is lethal, but co-overexpression of NQ1 partially rescues this lethality, resulting in Drosophila that survive until eclosion. Thus, in both the wild-type and overexpression contexts, a domain that forms heritable aggregates acts as a negative regulator of Pum activity.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||aggregate; GluRIIa; neuromuscular junction; prion; pumilio; synapse|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||24 September 2007|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||25 Oct 2007|
|Last Modified:||26 Dec 2012 03:16|
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