Nab3’s localization to a nuclear granule in response to nutrient deprivation is determined by its essential prion-like domain

Abstract

Ribonucleoprotein (RNP) granules are higher order assemblies of RNA, RNA-binding proteins, and other proteins, that regulate the transcriptome and protect RNAs from environmental challenge. There is a diverse range of RNP granules, many cytoplasmic, which provide various levels of regulation of RNA metabolism. Here we present evidence that the yeast transcription termination factor, Nab3, is targeted to intranuclear granules in response to glucose starvation by Nab3’s proline/glutamine-rich, prion-like domain (PrLD) which can assemble into amyloid in vitro. Localization to the granule is reversible and sensitive to the chemical probe 1,6 hexanediol suggesting condensation is driven by phase separation. Nab3’s RNA recognition motif is also required for localization as seen for other PrLD-containing RNA-binding proteins that phase separate. Although the PrLD is necessary, it is not sufficient to localize to the granule. A heterologous PrLD that functionally replaces Nab3’s essential PrLD, directed localization to the nuclear granule, however a chimeric Nab3 molecule with a heterologous PrLD that cannot restore termination function or viability, does not form granules. The Nab3 nuclear granule shows properties similar to well characterized cytoplasmic compartments formed by phase separation, suggesting that, as seen for other elements of the transcription machinery, termination factor condensation is functionally important.

Discovery of Arginine Methylation, Phosphorylation, and Their Co-occurrence in Condensate-Associated Proteins in Saccharomyces cerevisiae

“…These included but were not limited to Ede1 (at S977), Nab3 (at S8), Pab1 (at S28 and S32), Rnq1 (at S173), Scp160 (at T24), Whi3 (at S33), and Nsp1 (at S729). These are all known to either undergo phase separation themselves or to modulate the phase separation behavior of other proteins. − We expected that many of our new sites had been missed previously due to incomplete analysis of proteins digested solely with trypsin. Consistent with this, we note that many new phosphosites would be on large tryptic peptides, outside of the mass range used for routine MS/MS analysis.…”

An Algorithm to Quantify Inducible Protein Condensates In Eukaryotic Cells

“…This dynamic re-localization is accompanied by a change in the RNA-binding profiles of these termination factors (Bresson et al, 2017;Creamer et al, 2011;Jamonnak et al, 2011). The initial finding of this redistribution and a more recent confirmation using confocal microscopy (Loya et al, 2018), employed manual counting to quantify the fraction of cells bearing the Nab3-Nrd1 granule. Our previous work also used a computational approach to detect granule formation over time in single cell time-lapse microscopy.…”

“…When starved cells are refed, the granules rapidly resolve and cell division resumes (Loya et al, 2018). The different extents of granule formation between strains led us to consider if they might emerge from starvation at distinct rates.…”

“…We utilized the essential S. cerevisiae RNA-binding transcription factors, Nab3 and Nrd1, as a paradigm to demonstrate the capabilities of a granule-calling algorithm. Nab3 and Nrd1 are RNA-binding proteins that contain glutamine-rich low complexity domains (Alberti et al, 2009;Loya et al, 2013b;Loya et al, 2018) as well as an RNA recognition motif, which mediates RNA-binding. Through binding to each other, RNA, and RNA polymerase II-binding, they mediate the termination of RNA polymerase II transcription for many yeast transcripts, particularly small noncoding RNAs such as snRNAs and snoRNAs, as well as select mRNAs (Arndt and Reines, 2015;Creamer et al, 2011;Jamonnak et al, 2011;Jenks et al, 2008;Kopcewicz et al, 2007;Loya et al, 2013a).…”

“…Nab3 and Nrd1 are excellent candidates for studying protein rearrangement as these proteins reorganize into tight puncta in nuclei when cells are deprived of glucose as a carbon source (Darby et al, 2012). Assembly requires the Nab3 LCD and is rapidly and fully reversible when cells are fed metabolizable sugar (Loya et al, 2018). This punctate structure is novel as Nab3 does not colocalize with the stress granule marker Pub1, the P-body marker Dcp2, or the nucleolar marker Nop1, (Darby et al, 2012) making it a new class of subcellular structure that contains transcription termination factors but whose function is poorly understood.…”