cytoplasmic degradation of splice- defective pre-mrnas and intermediates patricia j. hilleren and...
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Cytoplasmic Degradation of Splice-Defective Pre-mRNAs and
IntermediatesPatricia J. Hilleren and Roy Parker
Hilleren et al., Mol. Cell 2003 Charu Shukla9.30.08
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Steady state level of a pre-mRNA depends on:• Rate of synthesis• Rate of splicing• Rate of degradationObserved changes in steady state levels of splice
defective RNA are assumed to result from a direct change in decay rates.
No direct measurement of the decay rates have been reported.
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Reporter system to measure decay rates of defective pre mRNA
• Combination of pulse chase experiments and the measurement of decay from steady state.
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GAL-mPGK1pG-WT (pRP1096)
ANALYSIS OF Pre-mRNA DEGRADATION
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GAL-mPGK1pG-C1 GAL-mPGK1pG-A257
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THE C1 and A257 Pre-mRNA DEGRADE VIA CYTOPLASMIC 5’ TO 3’ mRNA TURNOVER PATHWAY
Dcp1/Dcp2p- Decapping enzymes, initiate 5’ to 3’ decay pathway
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Xrn1p- 5’ to 3’ exoribonuclease (cytoplasm)Rat1p- 5’ to 3’ exonuclease (nucleus)
DECAY FROM STEADY STATE EXPERIMENTS
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• Pre mRNA that fail to assemble spliceosomes are exported and degraded by the cytoplasmic 5’ to 3’ mRNA turnover machinery.
• What about the nuclear exosome components??
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TRANSCRIPTION PULSE-CHASE ANALYSIS OF C303 REPORTER RNA
Rrp6p- nuclear exosome cofactor1. Spicing is blocked2. Lariat intermediate following the pulse degrades over time 5’ to 3’
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Rrp44-1-temp sensitive lesion of core exosome component Rrp44p
Dbr1p- endoribonuclease that cleaves 2’ to 5’ phosphodiester bondsResponsible for clearing the lariat byproduct of splicing
Degradation of lariat-exon2 splice defective intermediate is NOT dependent on nuclear exosomeRole of Dbr1p in initiating degradation.
Novel 3’ trimmedSpecies
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Rat1p-Nuclear exonuclease (5’ to 3’)Xrn1p-cytoplasmic exonuclease (5’ to 3’)
Lariat intermediate degrades by debranching followed by degradation by Xrnp1 and/or Ski2p
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3’ trimmed species
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3’ trimmed species
Abundance of C303 lariat intermediates and decay intermediates using P32-labelled oRP141
Reprobe to detect 7sRNA levels
In the absence of debranching, cytoplasmic exosome degrades the lariat intermediate.
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Dbr1p FUNCTIONS IN QUALITY CONTROL DURING PRE mRNA SPLICING
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MODEL FOR KINETIC COMPETITION AMONG PROCESSES THAT IMPACT FATES OF YEAST PRE mRNA
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Conclusions
• Pre mRNA not associating with the spliceosomes are degraded in the cytoplasm.
• Cytoplasmic pathway of degradation for lariat intermediates:
Debranching (Dbr1p) followed by 5’ to 3’ degradation (Xrn1p)
Debranching blocked: 3’ to 5’ degradationNo role of nuclear exosome in metabolism,
stability or abundance of C303 lariat intermediate