The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3' processing pathway

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• The nuclear exosome, particularly Rrp6, plays a crucial role in RNA processing and degradation.
• Cytoplasmic mRNA stability is regulated by ARE-binding proteins like Cth2.
• Understanding mRNA processing pathways is essential for deciphering gene expression control.

Purpose of the Study:

• To investigate the processing of CTH2 mRNA, an ARE-binding protein transcript.
• To elucidate the role of the nuclear exosome and associated factors in CTH2 mRNA maturation.
• To identify the regulatory mechanisms governing CTH2 mRNA levels.

Main Methods:

• Microarray analysis to identify over-expressed genes in exosome mutants.
• Northern blot analysis to detect and quantify specific RNA species.
• RNA 3' end mapping to determine transcript termini.
• Genetic analysis using mutant strains deficient in specific RNA processing factors (e.g., Rrp6, TRAMP4, Nrd1/Nab3/Sen1, Rat1, polyadenylation factors).

Main Results:

• Microarray analysis identified CTH2 mRNA as over-expressed in Rrp6-deficient strains.
• Exosome mutants accumulate a 3'-extended CTH2 precursor transcript instead of mature mRNA.
• The 3' end of CTH2 mRNA is mapped to a novel [GU(3-5)](5) repeat.
• CTH2 mRNA processing is independent of canonical polyadenylation factors (Rna14, Rna15, Pap1) but dependent on the TRAMP4 and Nrd1/Nab3/Sen1 complexes.
• The 3'-extended CTH2 pre-mRNA contains Nrd1 and Nab3 binding sites.
• Mutation of the ARE element or inactivation of Rat1 leads to elevated pre-mRNA and mRNA levels.

Conclusions:

• CTH2 mRNA is processed from a 3'-extended primary transcript through an unconventional pathway involving the exosome, TRAMP, and Nrd1/Nab3/Sen1 complexes.
• This pathway facilitates nuclear, ARE-mediated regulation of CTH2 mRNA levels, involving the nuclear 5'-exonuclease Rat1.
• The findings reveal a novel mechanism for regulating gene expression through pre-mRNA processing and nuclear-based regulatory elements.