Accumulation of CTH2 mRNA does not require the normal mRNA 3′ cleavage and polyadenylation machinery and the mRNA has an unusual site of polyadenylation. (A) Effect of ts-lethal mutations of the cleavage and polyadenylation machinery on CTH2 mRNA accumulation. RNA samples from indicated strains grown at 23°C and then shifted at 37°C for 1 h were analysed for CTH2, RPL30 mRNA expression and SCR1 as a loading control by northern blot. (B) CTH2 species present in rna15-2, rna14-1 and pap1-5 mutants at non-permissive temperature are deadenylated. Total RNA extracts of rna15-2, rna14-1 and pap1-5 mutants, grown at non-permissive temperature (37°C) for 1 h, were digested by RNase H in presence of an oligonucleotide located 50 nt before the estimated poly(A) site and with or without an oligo dT. Poly(A)⁺ and poly(A)^– species are indicated on the right of the panel. (C) The poly(A) tail is added on the CTH2 mRNA at a non-conventional poly(A) site. 3′-end RACE was performed on total RNA extracted from the wild-type strain. The poly(A) sites identified were located in the [GU_3–5]₅ region and are indicated by arrows.

Inactivation of the exosome or TRAMP complex induces accumulation of 3′ extended CTH2 mRNA. (A) Northern-blot analysis for CTH2 mRNA expression of total RNA samples extracted from wild-type, rrp6Δ, air1Δ+air2Δ, trf4Δ and trf5Δ strains grown at 23°C. The 3′-extended transcript is labelled pre-CTH2. (B) Northern-blot analysis of RNA samples extracted from wild-type strain grown in glucose, GAL::rrp41 and GAL::mtr4 strains pre-grown in galactose (GAL) and then shifted to glucose medium for 20 h (GLU). (C) Northern-blot analysis of RNA extracts from isogenic wild-type and ski7Δ mutant grown at permissive temperature (23°C lanes) and then shifted for 1 h at non-permissive temperature (37°C lanes). (D) Characterization of the 3′-extended transcript. 3′-end RACE was performed on total RNA extracted from the trf4Δ strain. The poly(A) tails in nine clones sequenced were located around 1850 nt after the stop codon (illustrated with arrows on the figure), while the remaining clone was located 120 nt closer to the CTH2 ORF.

CTH2 mRNA expression is regulated by ARE-dependent degradation and by the nuclear-specific 5′-exonuclease Rat1. (A) Half-life analysis of the mini-PGK1-CTH2 reporter plasmid with or without mutation of the ARE element. Wild-type strains were transfected with Mini-PGK1-CTH2 plasmid (Figure 4C) containing either the wild-type ARE element (WT) or a double-point mutated ARE element (ΔARE). Half-lives of reporters (WT and ΔARE) have been determined by northern blot analysis of total RNA extracted from cultures harvested at different time points after addition of doxycycline (2 µg/ml) to inhibit transcription. (B) Quantification of half-live measurements. The graph shows mean values ± standard deviations for the mini-PGK1-CTH2 transcript, obtained from quantification of three independent experiments and normalized to the SCR1 loading control. (C) Regulation of CTH2 mRNA expression by Rat1. Northern blot analysis of total RNA extracted from isogenic wild-type and rat1-2 strains at permissive temperature (23°C) and 30 min, 1 h and 2 h after transfer to non-permissive temperature (37°C). (D) Quantification of the previous experiment. The graph shows mean values ± standard deviations for the endogenous CTH2 transcript, obtained from quantification of three independent experiments and normalized to the SCR1 loading control.

Extended Cth2 transcripts are detected in wild type and trf4 strains. (A) Locations of PCR primers for detection of 3′ extended CTH2 transcripts. (B) Some CTH2 mRNA is not cleaved co-transcriptionally. RT–PCR reactions were performed on RNA samples from wild-type, trf4Δ and cth2Δ strains with PCR primers located on either side of the CTH2 poly(A) site, and separated on an agarose gel.

Mutation of Nrd1, Nab3 or Sen1 induces accumulation of 3′ extended CTH2 mRNA. (A) Accumulation of 3′ extended transcript of Cth2 in mutants of Nrd1/Nab3 complex. Northern-blot analysis of RNA extracts from wild-type, nrd1-102 and nab3-11 mutants grown at permissive temperature (23°C lanes) and then shifted for 1 h at non-permissive temperature (37°C lanes). The 3′-extended transcript is labelled pre-CTH2 and the asterisk indicates a putative processing intermediate. The asterisk marks a species 150–200 nt longer than the mature mRNA. (B) Accumulation of 3′-extended CTH2 in strain carrying a mutation in SEN1. Northern blot analysis of RNA extracts from isogenic wild-type and sen1-1 mutant grown at permissive temperature (23°C lanes) and then shifted for 1 h at non-permissive temperature (37°C lanes). (C) Representation of the mini-PGK1-CTH2 reporter. The reporter is under the control of a tet_off promoter (tetO₇ Pr), followed by the mini-PGK1 ORF sequence (Mini-PGK1), a poly(G) cassette (G₁₈) followed by the full-length CTH2 3′-UTR (2000-nt downstream of the CTH2 ORF). (D) Accumulation of a mini-PGK1-CTH2 extended transcript in strains carrying mutation in Nrd1 and Nab3. Northern blot analysis of the Mini-PGK1-CTH2 reporter expression in from wild-type, nrd1-102 and nab3-11 mutants grown at permissive temperature (23°C lanes) and then shifted for 1 h at non-permissive temperature (37°C lanes).

Mature Cth2 mRNA processing model. Data presented here suggest that CTH2 mRNA transcription goes far beyond the poly(A) signal and that co-transcriptional cleavage at the poly(A) site does not occur. Binding of the Nrd1/Nab3 complex onto the transcript, in association with the action of Sen1, might terminate transcription and/or recruit TRAMP and the exosome complexes onto the transcript. With the help of the TRAMP complex, the exosome is then able to degrade the primary transcript but pauses at the [GU_n]₅ site, where the transcript is subsequently polyadenylated by Pap1.