Yeast Core Exosome and Rrp44 Functions
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Questions and Answers

What is the purpose of the C-terminal tag in the Rrp44 expression plasmids?

  • To alter cell membrane permeability
  • To increase plasmid replication
  • To facilitate purification via streptavidin (correct)
  • To enhance protein stability

Which buffer was used for exosome purification during the experiments?

  • MgCl2 buffer
  • PBS buffer
  • TMN150 buffer (correct)
  • TE buffer

What method was used to create point mutations in the Rrp44 expression plasmids?

  • CRISPR technique
  • PCR amplification
  • QuikChange kit (correct)
  • Site-directed mutagenesis

What was the method used to normalize enzyme concentrations during the experiments?

<p>Immunoblotting (B)</p> Signup and view all the answers

What effect does treating the samples with 800 mM MgCl2 have during the purification process?

<p>It dissociates endogenous Rrp44 (A)</p> Signup and view all the answers

What is the dual function of the C-terminal S1 RNA-binding domain?

<p>Nuclease and structural element (A)</p> Signup and view all the answers

What is the consequence of a mutation like G916E on the exosome complex?

<p>Impairment of activity in vitro and in vivo (D)</p> Signup and view all the answers

Which of the following statements is true about Rrp44?

<p>Rrp44 can be substituted by other nucleases in the nucleus. (B)</p> Signup and view all the answers

What happens if a single core component of the exosome is depleted?

<p>It is essential for cell survival. (B)</p> Signup and view all the answers

What is the importance of the exosome complex?

<p>It is critical for RNA processing and degradation. (A)</p> Signup and view all the answers

Which technique was used to analyze the growth of yeast strains expressing Rrp44?

<p>Growth analysis in selective medium (D)</p> Signup and view all the answers

What does the presence of the GAL::rrp44 strain suggest about Rrp44's role?

<p>Its expression is regulated by glucose levels. (B)</p> Signup and view all the answers

What was utilized for the visualization of specific RNA sequences in the study?

<p>Northern hybridization (D)</p> Signup and view all the answers

What was the outcome of transferring the GAL::rrp44 strain to glucose medium?

<p>Growth was fully restored by the expression of Rrp44. (C)</p> Signup and view all the answers

What effect does deletion of the RRP44 ORF have on its function?

<p>It has no effect on the essential functions of Rrp44. (C)</p> Signup and view all the answers

How did the doubling time of the rrp44D strains compare when complemented by Rrp44-exo and WT Rrp44?

<p>Rrp44-exo had a doubling time of 3 hours, while WT Rrp44 had 2 hours. (A)</p> Signup and view all the answers

What characteristic defect is associated with genetic depletion of Rrp44?

<p>Decreased maturation of the 5.8S rRNA. (A)</p> Signup and view all the answers

What was indicated about Rrp44's exonuclease activity based on the study?

<p>It is redundant with other nucleases. (A)</p> Signup and view all the answers

Which plasmid resistance marker was used when constructing the rrp44D strain?

<p>URA3 (C)</p> Signup and view all the answers

What happens to cells expressing Ura3 when they lose the URA3 plasmid?

<p>They are killed by 5-FOA treatment. (C)</p> Signup and view all the answers

What significant result was observed when comparing strains expressing only Rrp44-exo to those expressing WT Rrp44?

<p>Rrp44-exo had a significantly longer doubling time than WT Rrp44. (C)</p> Signup and view all the answers

What mutation is associated with Rrp44 that allows it to generate degradation intermediates?

<p>Rrp44-exo (A)</p> Signup and view all the answers

What is suggested about the degradation activity of Rrp44-exo in the presence of Mn2+ ions?

<p>It significantly enhances degradation activity. (B)</p> Signup and view all the answers

What aspect differentiates Ssd1-v from Ssd1-d in yeast strains?

<p>Ssd1-v is the full-length protein, while Ssd1-d is truncated. (A)</p> Signup and view all the answers

What does the Rrp44-exo mutation paired with point mutations at conserved active-site amino acids create?

<p>Rrp44-endo-exo (C)</p> Signup and view all the answers

What effect did the depletion of intact Rrp44 have on the growth of W303 strains?

<p>It caused slightly less efficient growth. (A)</p> Signup and view all the answers

How was RNA degradation monitored in the study?

<p>By generating a ladder of degradation intermediates. (D)</p> Signup and view all the answers

What is the role of the PIN domain in the context of Rrp44 activity?

<p>It is associated with endonuclease activity. (A)</p> Signup and view all the answers

What does the genetic interaction of Ssd1 indicate about its function?

<p>It plays a role in RNA turnover and/or surveillance. (B)</p> Signup and view all the answers

What role do the alterations in the C-terminal regions of Rrp44 play concerning the exosome?

<p>They regulate exonuclease activity. (D)</p> Signup and view all the answers

Which of the following conclusions can be drawn about Rrp6 and Rrp44 interactions?

<p>They are important only for a subset of Rrp6 substrates. (C)</p> Signup and view all the answers

What distinguishes the functions of eukaryotic Rrp44 from bacterial RNase R?

<p>Rrp44 forms part of a redundant network of 30-exonucleases. (B)</p> Signup and view all the answers

Which of the following is a characteristic of the eukaryotic exosome?

<p>It possesses both 30-exonuclease and endonuclease activity. (C)</p> Signup and view all the answers

What activity does the PIN domain of Rrp44 appear to link?

<p>It links Rrp44 to the rest of the exosome. (C)</p> Signup and view all the answers

In E. coli, what does PNPase associate with in the degradosome complex?

<p>With RNase E and other proteins. (B)</p> Signup and view all the answers

What influence does the balance between RNA processing and decay have?

<p>It is more crucial in the cytoplasm than in the nucleus. (B)</p> Signup and view all the answers

What significant role do the proteins of the Rex family play within the exosome context?

<p>They fine-tune the network of 30-exonucleases. (C)</p> Signup and view all the answers

What role does the SKI2 DEVH box protein have in yeast?

<p>Facilitation of DNA manipulation (A)</p> Signup and view all the answers

Which element is associated with the stable maintenance of chromosomes in yeast?

<p>SSD1/SRK1/SSL1 gene (C)</p> Signup and view all the answers

What is the primary use of the versatile toolbox for PCR-based tagging in yeast genes?

<p>To allow gene deletion and modification (B)</p> Signup and view all the answers

Which protein complex is linked to pre-mRNA splicing and identified as a general splicing mutant suppressor?

<p>SSD1/SRK1 (B)</p> Signup and view all the answers

What type of activity is enhanced by Dis3 in relation to RCC1?

<p>GEF activity (C)</p> Signup and view all the answers

Which process does the 30 to 50 exonucleases involve in yeast according to the context provided?

<p>RNA degradation (A)</p> Signup and view all the answers

What is the function of the additional modules mentioned for PCR-based gene manipulation?

<p>To provide more tagging options (A)</p> Signup and view all the answers

What is one role associated with the RNA binding protein Ssd1p in yeast?

<p>Localization of proteins (B)</p> Signup and view all the answers

Flashcards

Exosome complex function

The exosome complex plays a role in RNA processing and degradation.

Exosome core components

Critical parts of the exosome complex, whose absence is lethal.

Rrp44 exonuclease activity

Rrp44's ability to break down RNA; important for the exosome but can be missing from the organism.

Yeast strain viability

Relates to whether yeast strains with specific characteristics (like missing Rrp44's exonuclease activity) can survive.

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Rrp44 mutation effect

Mutations in Rrp44 can impact substrate binding and the exosome's activity, possibly causing problems in the system.

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S1 RNA-binding domain

Part of a protein (Rrp44) that binds S1 RNA sequences, essential for exosome interaction.

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Core exosome association

Specific parts of a protein, like Rrp44, that bind with the exosome complex.

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Substrate binding and activity

How well a protein binds to and acts on its target molecule.

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Rrp44 expression plasmids

Yeast expression plasmids that carry the gene for Rrp44, a protein.

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TEV cleavage site

A specific sequence in a protein that allows the removal of a tag.

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Csl4-TAP strains

Yeast strains used for isolating complexes, grown in YPD medium.

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Exosomes purification buffer

Buffer TMN150 (Tris-HCl, NaCl, NP-40, MgCl2) used to purify exosomes.

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GST pull down assays

Processes used to identify protein-protein interactions.

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Rrp44 function in vivo

Rrp44's essential function in living cells doesn't require its exonuclease activity.

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Rrp44-exo

A catalytically inactive version of Rrp44.

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GAL::rrp44 strain

A strain engineered to express Rrp44 under the GAL promoter.

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Genetic depletion of Rrp44

Removing Rrp44 from a cell's genetic makeup.

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Exosome component defects

Problems in the 30-maturation process of 5.8s rRNA and degradation of the 50 ETS.

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5' -ETS

The portion of the Pre-rRNA prior to the 5S rRNA.

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Reduced growth rate

Cells' growth rate slows down.

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Doubling time

The time it takes for a population to double in size.

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What's the function of Ssd1 in yeast?

Ssd1 is a protein involved in RNA turnover and surveillance in yeast.

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Ssd1 variants in yeast

Yeast strains can have different forms of Ssd1. Some strains have the full-length version (Ssd1-v), while others have a truncated version (Ssd1-d).

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Rrp44-exo mutant's activity

The Rrp44-exo mutant lacks the exonuclease activity but can still generate degradation intermediates.

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Rrp44-endo mutant

This mutant has mutations in the active site of the PIN domain, affecting its endonuclease activity.

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Rrp44-exo-S1 mutant

This mutant has a mutation in the S1 RNA-binding domain, affecting how Rrp44 interacts with RNA.

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Strain differences in Rrp44 function

The activity of Rrp44 can be affected by the strain background. Some strains show better growth when Rrp44 is intact.

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Importance of Ssd1 for Rrp44 activity?

While Ssd1 is involved in RNA turnover, it's not clear if it directly affects Rrp44's activity.

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Exosome's Role

The exosome complex is involved in both RNA processing (modifying RNA) and degradation (breaking down RNA).

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Exosome's Location

The exosome operates in both the nucleus (where DNA is) and the cytoplasm (the cell's main space) but with different functions.

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Rrp44's Dual Role

Rrp44 is a protein that connects to the exosome core and has its own exonuclease activity, breaking down RNA.

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Rrp6 & Rrp44's Collaboration

Rrp6 and Rrp44 work together to degrade some RNA molecules, but can also act independently depending on the target.

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Rrp44's C-terminal Region

The C-terminal region of Rrp44 interacts with other exosome proteins and likely controls its exonuclease activity.

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Exosome and PNPase

The exosome's core structure resembles that of bacterial PNPase, which is involved in RNA degradation.

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Redundant Exonuclease Network

Multiple exonucleases (like Rrp44, Rrp6, and Rex family) work together to degrade RNA ensuring precise regulation.

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Exosome Complex

A group of proteins in yeast cells responsible for breaking down RNA molecules.

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Exonuclease

An enzyme that breaks down RNA by chewing away at the ends.

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SKI2 DEVH

A protein that helps the exosome complex work properly, specifically in RNA degradation.

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Yeast Strains

Different versions of yeast cells that vary in their genes and characteristics.

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DNA Manipulation

The process of changing or modifying DNA sequences in yeast.

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PCR-based Gene Deletion/Modification

A technique using polymerase chain reaction to remove or change genes in yeast.

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Fluorescent Proteins

Proteins that glow under certain light, helping track where other proteins are in cells.

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Protein Interaction Networks

The complex web of connections between proteins inside cells.

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Study Notes

Yeast Core Exosome and Rrp44

  • Yeast exosome components are shared between nuclear and cytoplasmic forms.
  • Rrp44 (Dis3) is a key 3'-5' exonuclease in the yeast core exosome.
  • Rrp44 lacking exonuclease activity (Rrp44-exo) supports growth.
  • Rrp44-exo is synthetic-lethal with loss of cytoplasmic 5'-exonuclease Xrn1, but not with loss of nuclear 3'-exonuclease Rrp6.
  • Rrp44-exo has a milder RNA processing phenotype than Rrp44 depletion.
  • Recombinant Rrp44 has manganese-dependent endonuclease activity.
  • The endonuclease activity is located in the N-terminal PIN domain.
  • Loss of exonuclease activity does not immediately cause synthetic lethality.
  • Loss of exonuclease activity of Rrp44 and Rrp6 results in reduced growth
  • Rrp44 is crucial for pre-rRNA maturation.
  • The exonuclease activity of Rrp44 is less critical for early steps in pre-rRNA processing.

Rrp44 Endonuclease Activity

  • Rrp44 has both exonuclease and endonuclease activity, depending on Mn2+ presence.
  • The N-terminal PIN domain is necessary and sufficient for exosome association.
  • Four conserved amino acids within the PIN domain bind Mn2+ and are essential for endonuclease activity.
  • The exonuclease activity is located in the RNB domain.
  • Rrp44-exo mutation shows no strong lethality with loss of Rrp6.
  • But Rrp44-exo is synthetically lethal with cytoplasmic 5'-exonuclease Xrn1.

Structural and Functional Role of Rrp44

  • The PIN domain of Rrp44 is required for exosome association.
  • The S1 RNA-binding domain is important for substrate binding and activity.
  • The presence of Mn2+ inhibits 3'-exonuclease activity but stimulates endonuclease activity.
  • Rrp44's N terminal PIN domain is vital for exosome core structure linking.
  • Rrp44-endo mutation maintains the endonuclease activity of Rrp44.
  • The exosome's exonuclease activity is critical in RNA processing.

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Related Documents

Rrp44 Harbors Endonuclease Activity (Nucleic Acids Research 2009) PDF

Description

Explore the intricate roles of Rrp44 in the yeast core exosome. This quiz delves into the exonuclease and endonuclease activities of Rrp44, their developmental impacts, and the synthetic lethality involved in its functions. Test your understanding of RNA processing and pre-rRNA maturation in yeast.

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