We find that the PPR1 mRNA is degraded 3-fold slower in upf1Δ yeast cells compared with UPF1 yeast cells (Fig. ) and that PPR1 mRNA steady-state levels are increased in upf2Δ, upf3Δ, dcp1Δ, xrn1Δ and hrp1-3 yeast cells (Figs and ). Thus, the factors required for the degradation of nonsense mRNAs by NMD are also required for the degradation of PPR1 mRNA. The PPR1 mRNA cis-acting sequence, called the UDE, maps to a region from the 5′-UTR to +92 in PPR1 mRNA (Figs –). This sequence targets PPR1 mRNA for accelerated decay by a novel mechanism (see below).
PPR1 mRNA is an unstable mRNA. Pierrat et al
) measured the half-lives of mRNAs encoded by chimeric genes constructed by swapping regions of PPR1
in wild-type cells to identify what parts of PPR1
are responsible for the rapid decay of PPR1 mRNA. They showed that the PPR1 5′-UTR contains a general instability element because it is sufficient to destabilize the URA3 mRNA. In contrast, UDE function requires sequences from both the 5′-UTR and the first 92 nt of the ORF. Thus, the general instability element may be distinct from the UDE. Alternatively, the UDE may have two parts, a general instability region and a region targeting the mRNA for Upf1p-dependent decay. The later possibility predicts that the general instability region functions independently, while the Upf1p-targeting region requires the presence of the general instability region to target PPR1 mRNA for Upf1p-dependent decay. We plan to test these possibilities by more precisely mapping the location of the UDE, and functional characterization and mutational analysis of the UDE.
Six wild-type mRNAs, PPR1, SPT10, UBP7, STE50 and CPA1 mRNAs, are degraded more rapidly in UPF1
yeast strains than upf
and this study). Wild-type mRNAs could potentially be subject to Upf-dependent decay either because cis
-elements trigger a premature translation termination event in an otherwise wild-type mRNA or by a novel mechanism. Premature translation termination can be triggered in an otherwise wild-type mRNA by uORF, extended 3′-UTRs, leaky scanning and potentially a ribosome frameshift.
mRNAs with artificially created uORFs are degraded by NMD (40
). However, natural uORFs do not necessarily target mRNAs for NMD. GCN4 and YAP1 mRNAs contain uORFs but are not subject to NMD-dependent decay (48
). The PPR1 and SPT10 uORFs do not target these mRNAs for Upf-dependent decay (39
). CPA1 mRNA also contains a uORF, but its role in the NMD-dependent decay of this mRNA has not been tested (47
mRNAs with abnormally long 3′-UTRs are also degraded by NMD (42
). A mutation in the CYC1
gene, deleting sequences required for proper 3′ end formation, results in the synthesis of mRNAs with abnormally extended 3′-UTRs ranging between 720 and 3970 nt. PPR1 mRNA does not appear to be targeted for Upf1p-dependent decay by an extended 3′-UTR for two reasons. First, the PPR1 mRNA 3′-UTR is within the normal range and considerably shorter than the 3′-UTRs resulting from deletion of the CYC1
3′ end processing signals. In yeast, most 3′-UTRs are ~100 nt in length and they can range in size from ~20 to ~350 nt (45
). The 3′-UTR of PPR1 is ~300 nt (43
). Secondly, the ACT1-PPR1
and PPR1 5
fusion constructs contain the PPR1
3′-UTR sequence and 3′ end processing signals (Fig. B, constructs 2 and 5). If an extended 3′-UTR targeted PPR1 mRNA for Upf1p-dependent decay, we would expect these constructs to encode mRNAs that are stabilized by inactivation of the NMD pathway. Because these mRNAs are not stabilized by inactivation of the NMD pathway, PPR1 mRNA is not targeted for Upf1p-dependent decay by an extended 3′-UTR (Fig. B, constructs 2 and 5).
Leaky scanning followed by translation initiation at an internal ORF triggers the NMD-dependent decay of SPT10 mRNA and probably UBP7, REV7 and STE50 (39
). These mRNAs share two key features: (i) a start codon in a sub-optimal context; (ii) an out-of-frame downstream AUG in an optimal context for translation initiation within 90 nt of the ORF start codon followed by an in-frame translation termination codon. The S.cerevisiae
optimal start codon context is ANNAUG
PuPuPu were N is any base and Pu is an A or a G. The PPR1 start codon context is ATCAUG
AAG, the optimal context. Further, there are no AUG codons within the following 90 nt. Therefore, the Upf1p-dependent decay of PPR1 mRNA is probably not due to leaky scanning.
The UDE may target PPR1 for Upf-dependent, deadenylation-independent decay either by triggering a ribosome frameshift or a novel mechanism. The PPR1 UDE could be a binding site for trans-acting factor(s) that target PPR1 for decay by interacting directly with the surveillance complex containing Upf1p, Upf2p and Upf3p. Alternatively, these trans-acting factors could induce the translation machinery to shift to a new reading frame. This would result in premature termination of translation and trigger NMD of PPR1 mRNA.
The UDE could be unique to PPR1 mRNA or alternatively it could be part of a family of similar sequences targeting wild-type mRNAs for NMD. We BLAST searched the S.cerevisiae DNA sequence datasets for similarities to the PPR1 UDE-region. No matches were found to sequences spanning both the 5′-UTR and the ORF (data not shown). This suggests that UDE is unique to PPR1 or similar elements are difficult to identify using this strategy because they have dispersed sequence elements or a degenerate sequence. Identification and analysis of the UDE sequence will be necessary to distinguish between these possibilities.
The PPR1 UDE could function alone as a destabilizing element or in conjunction with a DSE. Nonsense mRNAs require both the premature termination codon and a DSE to be recognized as a substrate for NMD. PGK1, ADE3 and HIS4 mRNAs all have DSEs with a loosely conserved motif (5′-TGYYGATGYYYYY-3′) (19
). There are four loose matches in PPR1 mRNA to the DSE consensus sequence. The potential matches (5′-TGCATGTAAACGATGT-3′, 5′-TGGCTGTCATGATGC-3′, 5′-TACGCCGATGT-3′ and 5′-TGCAAATAAATGATGC-3′) are located at +96, +251, +599 and +2249 in the PPR1 ORF. The first three of these potential DSEs are not essential for the Upf1p-dependent decay of PPR1 because when deleted, the resultant mRNA is still subject to Upf1p-dependent decay (See mini-PPR1
gene, Figs B construct 3 and B).
The accumulation of PPR1 mRNA in a hrp1-3
mutant at the non-permissive temperature provides indirect evidence that the putative DSE elements in PPR1 mRNA may be functional. Hrp1p specifically binds the PGK1 DSE and mutations in HRP1
stabilize nonsense mRNAs but not wild-type mRNAs (23
). The stabilization of PPR1 mRNA in a hrp1-3
mutant at the non-permissive temperature suggests Hrp1p also binds PPR1 mRNA, probably at one or more of the putative DSE elements. This supports the model in which the UDE functions in conjunction with a DSE. Characterization of the potential PPR1
DSE would add to our understanding of a class of Hrp1p-binding DSEs.