A nuclear surveillance pathway for mRNAs with defective polyadenylation - PubMed (original) (raw)

A nuclear surveillance pathway for mRNAs with defective polyadenylation

Laura Milligan et al. Mol Cell Biol. 2005 Nov.

Abstract

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3'-5' exonucleases, accumulation of poly(A)+ mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6delta. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3'-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation.

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Figures

FIG. 1.

FIG. 1.

The _pap1_-5, but not _pap1_-2, mutation allows poly(A) synthesis at the nonpermissive temperature and is suppressed by loss of Rrp6p. (A) Poly(A) tail length analysis of mRNAs from strains carrying the _pap1_-2 and _pap1_-5 mutations. Poly(A) tracts present in 7 μg of total RNA were labeled and analyzed on a 12% acrylamide-8 M urea gel. The size marker was tRNA from end-labeled total RNA. WT, wild type. (B and C) Northern blot analyses. The strains indicated were grown on glucose medium at 23°C (23°C lanes) and then shifted to 37°C for 2 h (37°C lanes). For each lane, 7 μg of total RNA was separated on a 1.2% agarose-formaldehyde gel and analyzed by Northern hybridization using the probes indicated on the left. The graphs show mean values ± standard deviations for the CYH2 transcript, obtained from PhosphorImager quantification of three independent experiments and normalized to the scR1 loading control.

FIG. 2.

FIG. 2.

Rrp6p acts prior to Rrp41p in the same degradation pathway. (A and B) Total RNA was extracted from the wild-type (WT), _pap1_-5, and _pap1_-_5/rrp6_Δ strains grown on glucose medium at 23°C and after shift to 37°C for the times indicated. Strains _pap1_-5/GAL::rrp41 and pap1_-5/rrp6_Δ/GAL::rrp41 were pregrown in galactose medium at 23°C (GAL lanes), transferred to glucose medium at 23°C for 20 h (23°C lanes), and then shifted to 37°C for the times indicated. Northern blot analysis was performed on 7 μg of total RNA separated on a 6% acrylamide-8.3 M urea gel (A) or a 1.2% agarose-formaldehyde gel (B). The graph shows levels of the CYH2 transcripts obtained by PhosphorImager quantification of the data presented in panel A normalized to the scR1 loading control. Values obtained at 23°C were arbitrarily set as 1. (C) Northern blot of heat shock-inducible mRNA. The wild-type, _pap1_-5, and _pap1_-_5/rrp6_Δ strains were pregrown on glucose medium at 23°C. The _pap1_-5/GAL::rrp41 and pap1_-5/rrp6_Δ/GAL::rrp41 strains were pregrown in galactose medium at 23°C and transferred to glucose medium at 23°C for 20 h (23°C lanes). All strains were then shifted to 42°C for 15 min (42°C lanes), followed by transfer to 37°C for the times indicated.

FIG. 3.

FIG. 3.

Specific mRNAs in the _pap1_-5 strain are polyadenylated. (A) Total RNA was extracted from the wild-type (WT), _pap1_-5, and _pap1_-_5/rrp6_Δ strains and grown in glucose medium for 30 min after transfer to 37°C. The _pap1_-5/GAL::rrp41 strain was pregrown in galactose medium at 23°C, transferred to glucose medium at 23°C for 20 h, and then shifted to 37°C for 30 min. Samples were treated with RNase H plus oligo(dT) (+ lanes) and compared with untreated samples (− lanes). Samples were separated on a 6% acrylamide-8.3 M urea gel, transferred to nylon, and hybridized with RPL25, RPL30, and MFA2 probes. T, deadenylated and truncated species. (B) PhosphorImager quantification of data from panel A. Deadenylated RPL25 and RPL30 mRNA (oligo-dT + lanes) was quantified using a PhosphorImager and standardized to scR1 RNA.

FIG. 4.

FIG. 4.

3′ degradation requires Mtr3p and Mtr4p. (A and B) Total RNA was extracted from the wild-type (WT), _pap1_-5, _mtr3_-1, and _pap1_-_5/mtr3_-1 strains grown on glucose medium at 23°C and after shift to 37°C for the times indicated. Strains GAL::mtr4 and _pap1_-5/GAL::mtr4 were pregrown in galactose medium at 23°C (GAL lanes), transferred to glucose medium at 23°C for 20 h (23°C lanes), and then shifted to 37°C for the times indicated. Northern blot analysis was performed on 7 μg of total RNA separated on a 6% acrylamide-8.3 M urea gel (A) or a 1.2% agarose gel (B). The graph shows levels of the CYH2 transcripts obtained by PhosphorImager quantification of the data presented in panel A, normalized to the scR1 loading control. Values obtained at 23°C were arbitrarily set as 1.

FIG. 5.

FIG. 5.

mRNA levels in _pap1_-5 strains lacking the exonuclease activity of Rrp6p. RNA was extracted from the strains indicated growing at 23°C and 1 h after transfer to 37°C. (A) RNA separated on 8% polyacrylamide-urea gel. (B) RNA separated on 1.2% agarose gels.

FIG. 6.

FIG. 6.

Growth curves obtained following transfer to 37°C. Cells were pregrown in rich YPD medium at 23°C and transferred to 37°C at time zero. The cells were maintained in exponential growth by addition of prewarmed medium. OD600, optical density at 600 nm.

FIG. 7.

FIG. 7.

Model for the degradation of pre-mRNAs in _pap1_-5 strains. Several pre-mRNA 3′ cleavage and polyadenylation factors bind to the C-terminal domain (CTD) of RNA polymerase II prior to recognition of the target site on the nascent RNA transcript (reviewed in references and 38). In the strains expressing the partially defective Pap1-5p, cleavage and polyadenylation still occur, but pre-mRNA surveillance is triggered. We speculate that this is a consequence of slowed polyadenylation. Deadenylation of the pre-mRNA requires the nucleus-specific exonuclease Rrp6p. Subsequent degradation requires the nuclear exosome complex indicated by the symbols on the right, and the putative RNA helicase Mtr4p.

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