Biochemical & Genetic Analysis of Low Complexity Domains in RNA-binding protein biology

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Summary/Abstract Many proteins involved in gene expression contain a low complexity domain that itself lacks secondary structure but which can form intermolecular polymers known as amyloid, a stable structure that can organize into filaments and hydrogels. Some of the proteins that bear such intrinsically unstructured domains interact with RNA polymerase II or the RNA produced during transcription. Indeed, this structural feature is unusually over-represented in cellular RNA- binding proteins. A current hypothesis suggests that these low complexity domains are proteinaceous switches that, with their adjacent folded portions such as RNA recognition motifs, assemble into cellular compartments that handle and process RNA. Excellent examples include yeast Nab3 and Nrd1 which are factors needed for termination of short transcripts such as snRNAs and snoRNAs. Similarly, potential amyloid forming domains that couple mRNA polyadenylation to termination at the end of protein coding genes are seen in Pcf11, Hrp1, and Rat1. Low complexity self-assembling domains are causally implicated in human diseases, particularly neurodegenerative pathologies in which aberrant cellular inclusions are a common characteristic. A leading model is that the normal propensity for aggregation of RNA-binding proteins can go awry, leading to toxic oligomeric and polymeric assemblies that are associated with cellular damage. Yet we have a poor understanding of the normal reversible function of these domains and why their aggregation can become toxic. The long-term goal of the project is to understand what amyloid forming domains provide for the function of RNA binding proteins; particularly those involved in terminating transcription of RNA polymerase II. The working hypothesis is that conditional polymerization of these proteins is a key part of how they operate. One model is that these proteins enshroud nascent RNA in a specific manner for presentation to the enzymes of termination and processing such as helicases and nucleases; perhaps in the way nucleosomes package DNA. The focus of this work is on RNA- binding, transcription termination factors in yeast where their involvement in the termination of transcription is fairly well understood. All possess low complexity domains that can potentially oligomerize. The prototype is the yeast hnRNP-like protein Nab3 that bears a polyglutamine-rich, self-assembly domain essential for cell viability and important for transcription termination. This project tests the role of these domains in the transcription termination machinery with respect to: 1) their amyloid forming potential, 2) the need for them for cell viability, and 3) their requirement for termination. Success in these aims will lead to new mechanistic models of how these RNA-protein interactions lead to productive and accurate gene expression.