An mRNA Surveillance Mechanism That Eliminates Transcripts Lacking Termination Codons
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The nonstop-PGK1 construct was generated from WT-PGK1 [pRP469 (8)] by creating three point mutations using site-directed mutagenesis (Quik-Change Site-Directed Mutagenesis Kit Stratagene) which eliminated the bona fide termination codon and all in-frame termination codons in the 3′ UTR. The Ter-poly(A)-PGK1 construct was created from the nonstop-PGK1 construct using site-directed mutagenesis (Quik-Change Site-Directed Mutagenesis Kit Stratagene). All changes were confirmed by sequencing. Primer sequences are available upon request. The PTC(22)-PGK1 construct is described in (8) (pRP609).
Leeds P., Wood J. M., Lee B. S., Culbertson M. R., Mol. Cell. Biol. 12, 2165 (1992).
P. A. Frischmeyer H. C. Dietz data not shown.
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We performed 3′ rapid amplification of cDNA ends (RACE) using the Marathon cDNA Amplification Kit (Clontech) and the Advantage 2 Polymerase Mix (Clontech) according to the manufacturer's instructions. Poly(A) RNA isolated from cells (Oligotex mRNA midi kit Qiagen) transiently transfected with WT-βgluc or from a wild-type yeast strain carrying a plasmid expressing WT-PGK1 was used as template. A single RACE product was generated cloned (TOPO TA 2.1 kit Invitrogen) and sequenced.
To create the WT-βgluc minigene construct mouse genomic DNA was used as template for amplifying three different portions of the β-glucuronidase gene: from nucleotide 2313 to 3030 encompassing exon 1 and part of intron 1 (including the splice donor sequence) from nucleotide 11285 to 13555 encompassing part of intron 9 (including the splice acceptor sequence ) exon 10 and part of intron 10 (including the splice donor sequence) and from 13556 to 15813 which included the remainder of intron 10 exon 11 intron 11 and exon 12. All three fragments were TA cloned (Topo TA 2.1 kit Invitrogen) sequenced and then cloned into pZeoSV2 (Invitrogen). A single base-pair deletion corresponding to the gus mps allele was generated by site-directed mutagenesis (Quik-Change Site-Directed Mutagenesis Kit Stratagene) of the WT-βgluc construct to create nonsense-βgluc. Nonstop-βgluc was generated from WT-βgluc by two rounds of site-directed mutagenesis (Quik-Change Site-Directed Mutagenesis Kit Stratagene) which removed the bona fide termination codon and all in-frame termination codons in the 3′ UTR. Ter-poly(A)-βgluc was generated from nonstop-βgluc via a single point mutation which created a stop one codon upstream of the poly(A) tail. All changes were confirmed by sequencing. Primer sequences are available upon request.
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All PGK1 minigene constructs are under the control of the GAL1 upstream activation sequence (UAS). Cultures were grown to mid-log phase in synthetic complete media–uracil (SC-ura) containing 2% galactose (transcription on). Cells were pelleted and resuspended in SC-ura media. Glucose was then added (transcription off) to a final concentration of 2% and aliquots were removed at the indicated time points. Approximately 10 μg of total RNA isolated with the hot phenol method (4) was electrophoresed on a 1.2% agarose formaldehyde gel transferred to a nylon membrane (GeneScreen Plus NEN) and hybridized with a 23 bp oligonucleotide end-labeled radioactive probe that specifically detects the polyG tract in the 3′ UTR of the PGK1 transcripts (8). Half-lives were determined by plotting the percent of mRNA remaining versus time on a semi-log plot. All half-lives were determined at 25°C except for the ccr4Δ (performed at 30°C) and dcp1-2 [performed after shift to the nonpermissive temp (37°C) for 1 hour] experiments.
HeLa cells [maintained in DMEM with 10% fetal bovine serum (FBS)] were grown to ∼80 to 90% confluency trypsinized and resuspended in 300 μl of RPMI 1640 without FBS 10 mM glucose and 0.1 mM dithiothreitol (DTT). Plasmid DNA (10 μg) was added and cells were electroporated (Bio-Rad Gene Pulser II Electroporation System) at a voltage of 0.300 kV and a capacitance of 500 μF. Poly
(A) RNA (Oligotex midi kit Qiagen 2 μg) isolated 36 hours after transfection was separated on a 1.6% agarose formaldehyde gel transferred to a nylon membrane and hybridized with a polymerase chain reaction (PCR) product comprising exons 10 through 12 of the β-gluc gene that had been radioactively labeled by nick translation (Random Primed DNA Labeling Kit Boehringer Mannheim). The membrane was subsequently stripped with boiling 0.5% SDS and probed with radioactively labeled zeocin cDNA. Northern blot results were quantitated using an Instant Imager (Packard). For subcellular fractionation transiently transfected HeLa cells were trypsinized washed in cold 1× phosphate buffered saline and pelleted by centrifuging at 2040 g for 5 min at 4°C. Cells were then resuspended in 200 μl of 140 mM NaCl 1.5 mM MgCl 2 10 mM Tris-HCl (pH 8.6) 0.5% NP-40 and 1 mM DTT vortexed and incubated on ice for 5 min. Nuclei were pelleted by centrifuging at 12 000 g for 45 s at 4°C and subsequently separated from the aqueous (cytoplasmic) phase.
Plasmid pG-26 (25) containing the CBP1 gene under the control of the GAL promoter and the LEU2 selectable marker was introduced into wild-type and SKI7 -deleted strains. Yeast were grown in media lacking leucine and containing 2% galactose at 30°C to an OD 600 of 0.5. Cells were pelleted and resuspended in media lacking a carbon source. Glucose was then added to a final concentration of 2% at time 0 and time points were taken. RNA was extracted and analyzed by northern blotting with a labeled oligo probe oRP1067 (5′-CTCGGTCCTGTACCGAACGAGACGAGG-3′).
We thank N. Martin A. Atkin C. Dieckmann and M. Sands for the provision of valuable reagents. This work was supported by a grant from NIH (GM55239) (H.C.D.) the Howard Hughes Medical Institute (H.C.D R.P.) and the Medical Scientist Training Program (P.A.F.).