Control of NutrientSensitive Transcription Programs by the Unconventional Prefoldin URI

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Control of Nutrient-Sensitive Transcription
Programs by the Unconventional Prefoldin URI
By Matthis Gstaiger, Brian Luke, Daniel Hess,
Edward J. Oakeley, Christiane Wirebelauer, marc
Blondel, Marc Vigneron, Matthias Peter, Wilhelm
Krek
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Background

• The TOR (target of rapamycin) pathway plays a
  central role in integration and transduction of
  nutritional cues into a cell-growth and
  proliferative response.
• Nutrient rich sustained TOR activity cell
  growth
• Nutrient poor inhibition of TOR activity
  nutrient sensitive gene expression

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Background

• Prefoldins (PFDs) are members of a small
  molecular weight protein family
• PFDs are able to assemble into molecular
  chaperone complexes
• 2 classes ß and a

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Materials and Methods

• Identified STAP 1 (SKP2-associated alpha PFD 1)
  protein. Thought STAP 1 could be part of a
  protein complex

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Mass Spectrometric ID

• 4 proteins linked to transcription
• RBP5 URI
• On gel filtration, STAP 1 eluted as a single peak
  with URI

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Immunoprecipitation

• Depletion of URI codepleted STAP 1 and PFD4r.
  They are stoichiometrically associated with URI
• STAP 1 and URI are part of a single complex?

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• Recognized a homology between STAP 1 and the ORF
  YFL023w in yeast.
• Conceptual translation of YFL023w predicted a
  protein, identified as scUrip.
• Orthologs of YFL023w found in other organisms,
  including Homo sapiens
• The human ortholog is identical to URI. So, this
  polypeptide is referred to as URI/scUrip

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URI/scUrip deletion

• Deleted scUrip gene in yeast. Identified 39
  genes whose expression was consistently affected
  by loss of scUrip.
• Products of these genes function primarily in
  amino acid metabolism

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URI/scUrip deletion
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URI/scUrip deletion

• URI deletion also resulted in the down-regulation
  of genes that encode different tRNA species
• So, scUrip contributes to expression of RNA pol
  II and pol III transcripts

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URI/scUrip deletion

• Loss of scUrip caused cell elongation and agar
  penetration hallmarks of growth induced by
  nutrient limitations

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URI/scUrip deletion

• scUrip levels decreased when cells grown in
  a.a.-deprived medium or in low-quality nitrogen
  source, or when cells entered stationary phase.

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• Exposed yeast cells to rapamycin or a.a.
  starvation
• Majority of genes affected by loss of URI are
  genes that are transcriptional targets of a.a.
  starvation or rapamycin signals.

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• Used hierarchical cluster analysis to compare
  wild-type cells with cells without URI
• Loss of scUrip affected induction of
  rapamycin-sensitive genes. Affected induction
  and repression of genes in a.a. starved cells

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• Large of genes activated in URI deleted cells
  contained a consensus-binding site for Gcn4p, a
  transcription factor known to activate a.a.
  biosynthesis genes.
• These genes failed to be induced in double
  mutants, so Gcn4p plays a role in activation when
  scURip is absent

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• Gcn4p levels up-regulated in URI deleted cells,
  under conditions of a.a. sufficiency.

• scUrip may contribute to suppression of GCN4
  translation

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Human TOR Signaling

• Serum starvation of cells (HEK293) caused
  dephosphorylation of URI.
• Phosphorylation state of URI is dependent on
  signals that affect mTOR activity

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Human TOR Signaling

• Created HeLa cells silenced for URI expression by
  siRNA. Compared their transcriptional profiles
  in response to rapamycin.
• 28 genes had transcriptional patterns
  signficantly altered in URI-silenced HeLa cells.

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Results

• STAP 1 and URI reside in a single high-molecular
  weight complex
• scUrip contributes to the expression of RNA pol
  II and pol III transcripts, specifically ones
  involved in nutrient response pathways
• Function of scUrip is critical for
  transcriptional responses that originate from
  distinct nutrient signals

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Results

• scUrip may contribute to GCN4-dependent
  transcription programs under nutrient-rich
  conditions (inhibit them).
• Loss of scUrip triggers activation of genes under
  the control of Gcn4p and Gln3p or Gat1p
• Phosphorylation state of URI is dependent on
  signals that affect mTOR activity

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Discussion

• URI is part of a multiprotein complex in human
  cells
• URI appears to occupy a central role as a
  scaffolding protein, using its PFD-homology and
  RPB5-binding domains
• scUrip contributes to TOR-mediated suppression of
  a select class of genes (GCN4, GLN3, GAT1) under
  nutrient rich conditions.

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Discussion

• In mammals, URI is a phosphorylation target of
  the mTOR pathway and contributes to
  rapamycin-sensitive transcription (like it does
  in yeast)
• This implies a conserved role for URI proteins in
  TOR signaling
• Further study is warranted into the functions URI
  and other PFDs serve in human diseases in which
  TOR signaling is implicated

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Thank you