title: Amyloid-like assembly of the low complexity domain of yeast Nab3.
abstract: Termination of transcription of short non-coding RNAs is carried out in yeast by the Nab3-Nrd1-Sen1 complex. Nab3 and Nrd1 are hnRNP-like proteins that dimerize and bind RNA with sequence specificity. We show here that an essential region of Nab3 that is predicted to be prion-like based upon its sequence bias, formed amyloid-like filaments. A similar region from Nrd1 also assembled into filaments in vitro. The purified Nab3 domain formed a macroscopic gel whose lattice organization was observed by X-ray fiber diffraction. Filaments were resistant to dissociation in anionic detergent, bound the fluorescent dye thioflavin T, and showed a β-sheet rich structure by circular dichroism spectroscopy, similar to human amyloid β which served as a reference amyloid. A version of the Nab3 domain with a mutation that impairs its termination function, also formed fibers as observed by electron microscopy. Using a protein fragment interaction assay, the purified Nab3 domain was seen to interact with itself in living yeast. A similar observation was made for full length Nab3. These results suggest that the Nab3 and Nrd1 RNA-binding proteins can attain a complex polymeric form and raise the possibility that this property is important for organizing their functional state during termination. These findings are congruent with recent work showing that RNA binding proteins with low complexity domains form a dynamic subcellular matrix in which RNA metabolism takes place but can also aberrantly yield pathological aggregated particles.
authors.affiliation: a Department of Biochemistry ; Emory University School of Medicine ; Atlanta , GA USA ;
authors.forename: Thomas W
authors.initials: TW
authors.lastname: O'Rourke
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Loya
authors.forename: PamelaSara E
authors.initials: PE
authors.lastname: Head
authors.forename: John R
authors.initials: JR
authors.lastname: Horton
authors.forename: Daniel
authors.initials: D
authors.lastname: Reines
mesh_terms: D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002942:Circular Dichroism; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction
chemical_list: D016229:Amyloid beta-Peptides; C090564:NAB3 protein, S cerevisiae; C491211:NRD1 protein, S cerevisiae; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: Aβ, amyloid beta; BSA, bovine serum albumin; CPEB, cytoplasmic polyadenylation element binding protein; CTD, carboxy terminal domain; DHFR, dihydrofolate reductase; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; GFP, green fluorescent protein; HFIP, hexafluoroisopropanol; IPTG, isopropyl β-D-1-thiogalactopyranoside; PCR, polymerase chain reaction; RNA binding protein; RRM, RNA recognition motif; SDD-AGE, semi-denaturing detergent agarose gel electrophoresis; SDS, sodium dodecyl sulfate; TEV, tobacco etch virus; amyloid; fibril; hnRNP; hnRNP, heterogeneous nuclear ribonucleoprotein; transcription termination