A Network of Interdependent Molecular Interactions Describes a Higher Order Nrd1-Nab3 Complex Involved in Yeast Transcription Termination

Loya, Travis J.; O'Rourke, Thomas W.; Degtyareva, Natalya; Reines, Daniel

doi:10.1074/jbc.m113.516765

Cited by 20 publications

(46 citation statements)

References 41 publications

(85 reference statements)

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“…10 This biochemical data, combined with genetic data indicating that 2 different mutant versions of Nab3 can complement cell viability in trans, suggests that multiple copies of this protein work together during termination. 9 This extends a model postulating that multiple copies of Nab3 and Nrd1 operate together on a short nascent transcript bearing consensus sequences, while interacting with RNA polymerase II to provoke termination. 4 Some proteins have regions that are considered intrinsically disordered, meaning the polypeptide backbone populates a dynamic set of positions in space; generally lacking fixed secondary structural elements.…”

Section: Introductionsupporting

confidence: 71%

“…These features, and the heat stability and protease sensitivity of the recombinant protein purified from E. coli, suggest it is intrinsically unstructured and prion- like. 8,9 Although unstructured, prion-like proteins can change conformation and polymerize to form ordered aggregates in vitro and in vivo. 23 Although essential, the function of this portion of the protein has remained obscure.…”

Section: Resultsmentioning

confidence: 99%

“…The following findings make it likely that filament formation is important for Nab3s function as an RNA-binding protein in transcription termination: 1) prior genetic analysis has supported a model that the assembly of multiple copies of Nab3 onto transcripts in living cells is needed for cells to survive, 9 2) evidence presented here using an independent assay indicates that the Nab3 low complexity domain interacts with itself in living cells, as does intact Nab3, 3) the region of Nab3 that assembles into filaments is essential for cell viability, 9 the small size of the region limits the number of functions it can contain, increasing the likelihood that its essential function is related to its polymerization potential, and finally, 4) recent findings across species have revealed that RNA-binding proteins, many of which are hnRNPs and many of which contain low complexity regions, undergo reversible associations important for their functions and yield filaments and semi-solid gels in vitro. 15,16 As well, amyloid formation by prion-like proteins is becoming recognized as a functionally important state and it forms the basis of regulatory switches in diverse organisms.…”

Section: Discussionmentioning

confidence: 99%

“…Mutations in the low complexity domain of Nab3 have been identified that compromise its ability to support termination of short, non-coding transcripts. 5,9,10 One of these is a Leu to Ala substitution at position 800 ( Fig. 1) that shows a strong terminator override phenotype.…”

Section: Nab3mentioning

confidence: 99%

“…[5][6][7] This domain of Nab3 is made up of a low complexity amino acid sequence that is intrinsically unstructured and prion-like based on prediction algorithms and its demonstrated biochemical properties. 8,9 Nab3's very C-terminal residues also contain a short region of structural homology to human hnRNP-C that is predicted to be a-helical and can crosslink to itself (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

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Amyloid-like assembly of the low complexity domain of yeast Nab3

O'Rourke

Loya

Head

et al. 2015

Prion

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y These authors contributed equally to this work.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 prionlike 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 b-sheet rich structure by circular dichroism spectroscopy, similar to human amyloid b 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.

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Section: Introductionsupporting

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Nab3mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Amyloid-like assembly of the low complexity domain of yeast Nab3

O'Rourke

Loya

Head

et al. 2015

Prion

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Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis

et al. 2022

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Nab3 nuclear granule accumulation is driven by respiratory capacity

2022

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Numerous biological processes involve proteins capable of transiently assembling into subcellular compartments necessary for cellular functions. One process is the RNA polymerase II transcription cycle which involves initiation, elongation, co-transcriptional modification of nascent RNA, and termination. The essential yeast transcription termination factor Nab3 is required for termination of small non-coding RNAs and accumulates into a compact nuclear granule upon glucose removal. Nab3 nuclear granule accumulation varies in penetrance across yeast strains and a higher Nab3 granule accumulation phenotype is associated with petite strains, suggesting a possible ATP-dependent mechanism for granule disassembly. Here, we demonstrate the uncoupling of mitochondrial oxidative phosphorylation by drug treatment or deletions of nuclear-encoded ATP synthase subunit genes, were sufficient to increase Nab3 granule accumulation and led to an inability to proliferate during prolonged glucose deprivation, which requires respiration. Additionally, by enriching for respiration competent cells from a petite-prone strain, we generated a low granule-accumulating strain from a relatively high one, providing another link between respiratory competency and Nab3 granules.Consistent with the resulting idea that ATP is involved in granule accumulation, the addition of extracellular ATP to semi-permeabilized cells was sufficient to reduce Nab3 granule accumulation. Deleting the SKY1 gene, which encodes a kinase that phosphorylates nuclear SR repeat-containing proteins and is involved in efficient stress granule disassembly, also resulted in increased granule accumulation. This observation implicates Sky1 in Nab3 granule biogenesis. Taken together, these findings suggest there is normally an equilibrium between termination factor granule assembly and disassembly mediated by ATP-requiring nuclear machinery..

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A Network of Interdependent Molecular Interactions Describes a Higher Order Nrd1-Nab3 Complex Involved in Yeast Transcription Termination

Cited by 20 publications

References 41 publications

Amyloid-like assembly of the low complexity domain of yeast Nab3

Amyloid-like assembly of the low complexity domain of yeast Nab3

Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis

Nab3 nuclear granule accumulation is driven by respiratory capacity

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