Cytoplasmic Degradation of Splice-Defective Pre- mRNAs and Intermediates Patricia J. Hilleren and Roy Parker Hilleren et al., Mol. Cell 2003 Charu Shukla 9.30.08
Dec 19, 2015
Cytoplasmic Degradation of Splice-Defective Pre-mRNAs and
IntermediatesPatricia J. Hilleren and Roy Parker
Hilleren et al., Mol. Cell 2003 Charu Shukla9.30.08
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.
Reporter system to measure decay rates of defective pre mRNA
• Combination of pulse chase experiments and the measurement of decay from steady state.
GAL-mPGK1pG-WT (pRP1096)
ANALYSIS OF Pre-mRNA DEGRADATION
GAL-mPGK1pG-C1 GAL-mPGK1pG-A257
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
Xrn1p- 5’ to 3’ exoribonuclease (cytoplasm)Rat1p- 5’ to 3’ exonuclease (nucleus)
DECAY FROM STEADY STATE EXPERIMENTS
• 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??
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’
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
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
3’ trimmed species
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.
Dbr1p FUNCTIONS IN QUALITY CONTROL DURING PRE mRNA SPLICING
MODEL FOR KINETIC COMPETITION AMONG PROCESSES THAT IMPACT FATES OF YEAST PRE mRNA
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