Eukaryotic transcripts that contain premature stop codons are degraded by the system of nonsense-mediated mRNA decay (NMD). NMD in C. elegans requires functions of the seven smg genes. smg mutants were isolated in our lab as phenotypic suppressors of
unc-54(
r293).
unc-54(
r293) is a small deletion that removes the
unc-54 polyadenylation signal without affecting the
unc-54 open reading frame. We have shown that
r293 expresses an unusually large mRNA in which
unc-54 sequences are fused to sequences of the gene immediately adjacent to
unc-54. The adjacent gene, F32A7.6, is transcribed in the same relative orientation as
unc-54, and the bicistronic
r293 mRNA contains the complete open reading frames of both genes. Besides alleles of the seven smg genes, a number of intragenic revertants were isolated during
unc-54(
r293) suppressor screens. Such alleles are dominant and inseparable from
unc-54. We have characterized the DNA sequences and mRNAs expressed by nine intragenic revertants of
r293. Northern analysis shows that all nine alleles accumulate
unc-54 mRNAs that are nearly normal in size and abundance. Sequence analyses demonstrate that all six chemically-induced mutations are single G to A transitions located downstream of the
unc-54(
r293) termination codon and upstream of the F32A7.6 initiation codon. This single base change creates the sequence AATGAA, which serves as a new cleavage and polyadenylation signal. Revertant mRNA are cleaved and polyadenylated 17 nt after the base change. Each of the three
mut-5 - induced alleles contains Tc1 insertions located downstream of the
unc-54(
r293) termination codon. Revertant mRNAs are cleaved and polyadenylated 180 nt within Tc1. We interpret these results to indicate that sequences within F32A7.6 are sufficient to destabilize
unc-54 mRNA when placed downstream of the
unc-54 termination codon. Perhaps F32A7.6, like most open reading frames, contains sequences analogous to "downstream elements" (DSEs) of yeast, which have been shown to promote NMD. According to this model, mRNAs of the
unc-54(
r293) intragenic revertants are stable because their new polyadenylation signals eliminate F32A7.6 DSEs from the message.