Mutant LYS2 mRNAs retained and degraded in the nucleus of Saccharomyces cerevisiae

Abstract

We previously demonstrated that mRNAs retained in the nucleus of Saccharomyces cerevisiae are subjected to a degradation system-designated DRN (degradation of mRNA in the nucleus), that is diminished in cbc1-Δ or cbc2-Δ mutants lacking components of the cap-binding complex and in rrp6-Δ mutants lacking Rrp6p, a 3′ to 5′ nuclear exonuclease. Two mutants, lys2-187 and lys2-121, were uncovered by screening numerous lys2 mutants for suppression by cbc1-Δ and rrp6-Δ. Both mutants were identical and contained the two base changes, one of which formed a TGA nonsense codon. LYS2 mRNAs from the lys2-187 and related mutants were rapidly degraded, and the degradation was suppressed by cbc1-Δ and rrp6-Δ. The U1A-GFP imaging procedure was used to show that the lys2-187 mRNA was partially retained in the nucleus, explaining the susceptibility to DRN. The creation of several derivatives of lys2-187 by site-directed mutagenesis revealed that the in-frame TGA by itself was not responsible for the increased susceptibility to DRN. Thus, mRNAs susceptible to DRN can be formed by a 2-bp change. Furthermore, this “retention signal” causing susceptibility to DRN is lost by altering one of the base pairs, establishing that mRNAs susceptible and unsusceptible to DRN can be attributed to a single nucleotide in the proper context.