Nuclear mRNA surveillance in THO/sub2 mutants is triggered by inefficient polyadenylation

Area of Science:

• Molecular Biology
• Yeast Genetics
• RNA Metabolism

Background:

• The THO complex and Sub2p are crucial for messenger ribonucleoprotein (mRNP) maturation in yeast.
• Mutants lacking THO/Sub2 exhibit accelerated mRNA decay mediated by the nuclear RNA exosome.
• The precise mechanism underlying this accelerated decay has remained unclear.

Purpose of the Study:

• To elucidate the molecular basis for accelerated mRNA decay in yeast THO/Sub2 mutants.
• To investigate the role of mRNA 3' end processing in this surveillance pathway.
• To identify factors linking THO/Sub2 function to mRNA stability.

Main Methods:

• Genetic screening to identify interactions between THO/Sub2 mutants and mRNA 3' end processing mutants.
• Nuclear run-on assays to assess transcription termination.
• In vitro polyadenylation assays using mutant yeast extracts.
• Analysis of Fip1p protein levels and regulation by the ubiquitin/proteasome pathway.
• Investigating the role of the nuclear exosome components Rrp6p and Trf4p.

Main Results:

• Inefficient polyadenylation is identified as the primary cause of accelerated mRNA decay in THO/Sub2 mutants.
• THO/Sub2 mutants show genetic interactions with mRNA 3' end processing mutants and premature transcription termination.
• Pre-mRNA substrates are poorly polyadenylated and unstable in THO/Sub2 mutant extracts.
• Decreased polyadenylation correlates with Fip1p downregulation via the ubiquitin/proteasome pathway.
• Both polyadenylation defects and Fip1p instability are dependent on the nuclear exosome components Rrp6p and Trf4p.

Conclusions:

• Accelerated mRNA decay in yeast THO/Sub2 mutants is driven by inefficient polyadenylation.
• The nuclear exosome, through Rrp6p and Trf4p, regulates polyadenylation efficiency, potentially by controlling Fip1p stability.
• These findings suggest a direct link between mRNA 3' end processing regulation and mRNA surveillance mechanisms.