Linking mRNA Turnover and Translation: Assessing the Polyribosomal Association of mRNA Decay Factors and Degradative Intermediates

METHODS: A Companion to Methods in Enzymology 17, 28 –37 (1999) Article ID meth.1998.0704, available online at http://www.idealibrary.com on

Linking mRNA Turnover and Translation: Assessing the Polyribosomal Association of mRNA Decay Factors and Degradative Intermediates David A. Mangus and Allan Jacobson Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655– 0122

mRNA decay is a multistep process, often dependent on the active translation of an mRNA and on components of the translation apparatus. In Saccharomyces cerevisiae, several trans-acting factors required for mRNA decay associate with polyribosomes. We have explored the specificity of the interactions of these factors with polyribosomes, using sucrose gradient sedimentation analysis of the yeast UPF1 protein to test whether such interactions are altered when polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal nuclease, or shifting of cells containing a temperaturesensitive eIF3 mutation to the nonpermissive temperature. These experiments, as well as others assaying the strength of factor association in high-salt sucrose gradients, lead us to conclude that Upf1p is tightly bound to the smallest polyribosomes, but not to the 40S or 60S ribosomal subunits. Similar experimental approaches were used to determine whether mRNA decay initiates prior to mRNA release from polyribosomes. Using sucrose gradient fractionation and Northern blotting, we can detect the polysomal association of a PGK1 mRNA decay intermediate and conclude that mRNA decay commences while an mRNA is still being translated. © 1999 Academic Press

The rates with which mRNAs are degraded are important determinants of the levels of gene expression (1, 2). For most yeast mRNAs, these rates reflect the combined efficiencies of three sequential events: shortening of the poly(A) tail, removal of the 59 cap, and 59 3 39 exonucleolytic digestion of the body of the mRNA (3–6). The enzyme(s) responsible for poly(A) shortening have yet to be defined, but those that catalyze decapping and exonucleolytic decay have been 28

shown to be the products of the DCP1 and XRN1 genes, respectively (6, 7). Experiments in yeast and other systems have also demonstrated that the intrinsic half-life of an mRNA can be reduced markedly by premature translational termination (2, 8–12). Like wild-type transcripts, mRNAs that are subject to such “nonsense-mediated mRNA decay” are decapped by Dcp1p and degraded exonucleolytically by Xrn1p, but become substrates for these enzymes without prior poly(A) shortening (7, 9). Several additional trans-acting factors are required to promote the rapid decay of nonsensecontaining mRNAs. These include, but are not limited to, the cytoplasmic proteins Upf1p, Nmd2p (Upf2p), and Upf3p (8, 13–16). Upf1p and Upf3p each interact with Nmd2p (14, 17), but these interactions probably do not occur simultaneously since the respective cellular concentrations of these proteins differ greatly (Upf1p, ;1600 molecules/cell; Nmd2p, ;160 molecules/cell; Upf3p, ;30 molecules/ cell (D.A.M. and A.J., unpublished experiments; 18)). These observations, and genetic studies showing that deletion of any one, or all, of the UPF/NMD genes leads to the same extent of stabilization of nonsense-containing mRNAs, suggest that these factors function in a linear pathway. While this pathway clearly has a role in regulating the stability of nonsense-containing mRNAs, its cellular function may be broader. Mutations in UPF1, NMD2, and UPF3 also lead to increases in ribosomal frameshifting and nonsense codon suppression, suggesting that the respective gene products may also regulate translational fidelity (8, 16, 19; A. Maderazo and A.J., manuscript in preparation). Consistent with this notion are recent observations that Upf1p inter1046-2023/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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acts genetically and biochemically with the peptide release factors, Sup35p and Sup45p (20; F. He and A.J., unpublished experiments). The capacity of a premature nonsense codon to trigger rapid mRNA decay suggests that the ability to translate an mRNA is an important determinant of its stability. Additional evidence suggesting that mRNA decay and translation are tightly linked includes experiments showing that (a) drugs or mutations that inhibit translation elongation also inhibit mRNA decay (2, 21, 22); (b) translation through a cis-acting instability element in the coding region of the yeast MATa1 mRNA is required to destabilize that transcript (22, 23); (c) nonsense-containing mRNAs associate with polyribosomes prior to their degradation (24); and (d) factors required for decay are associated with polyribosomes (10, 12, 18, 25). In this paper, we describe methods for assessing the significance of the association of mRNA decay factors with polyribosomes. We show that a fraction of all of the factors assayed cosediment with polyribosomes and we describe methods to affirm the specificity of this interaction for one factor, Upf1p. These experiments demonstrate that the sedimentation of Upf1p is altered when polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal nuclease, or shifting of cells containing a temperaturesensitive eIF3 allele to a nonpermissive temperature. Upf1p does not appear to associate with either the 40S or 60S ribosomal subunits, but is tightly bound to the smallest polyribosomes (those mRNAs with one to three ribosomes). This observation suggests that Upf1p, and other factors required for nonsense-mediated mRNA decay, may be required early in the life of an mRNA to determine if it is competent for translation. A similar experimental approach was used to determine whether mRNA decay initiates while mRNA translation is still underway. Our demonstration of an association between a specific decay intermediate and polyribosomes suggests that this possibility is most likely.

DESCRIPTION OF THE METHOD Assessing Factor and mRNA Association with Polyribosomes Polyribosomes, multiple ribosomes translating a single mRNA, were first identified as the site of cellular protein synthesis almost 35 years ago (26 – 29). These structures are only marginally stable and

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their isolation requires careful lysis of the cell and specific buffer conditions to prevent their disruption. Here, we describe methods to prepare extracts from Saccharomyces cerevisiae, and to determine whether factors involved in mRNA decay, as well as degradative intermediates, are associated with the translation apparatus. In the experiments that follow, mRNA decay factors have been either overexpressed from high-copy plasmids that facilitate their detection (e.g., Fig. 1) or assayed at their normal cellular concentrations (e.g., Figs. 2 and 3). The original analytical tool, sucrose gradient sedimentation, is still the best method to isolate and characterize polyribosomes. The presence of factors specifically associated with the translation apparatus can be assessed by fractionating the gradients and subsequently analyzing the samples by Western blotting. For all of the factors assayed in this study, a fraction of the protein present in the cell cosedimented with polyribosomes (Fig. 1). This conclusion applied to a factor required for general mRNA decay (Xrn1p), as well as to those specifically required for nonsense-mediated mRNA decay (Upf1p, Nmd2p, and Upf3p). As a first approximation, the association of these proteins with polyribosomes appears to be specific since their sedimentation was coincident with Tcm1p (ribosomal protein L3), but separated from Pgk1p (a cytosolic protein not associated with the ribosome). A significant portion of the decay factors analyzed was also present in the lighter fractions of the gradient (Fig. 1, lanes 9 –13). This may represent a free pool of these factors and/or mRNPs that are not being translated. Several different tests can be used to determine the specificity of a factor’s apparent interaction with the translation apparatus. For simplicity, we present each test using only data from Upf1p experiments as illustrative examples. Since the formation and maintenance of 80S ribosomes requires the presence of Mg21 ions, polyribosomes can be disrupted by adding EDTA to the extracts. Samples treated this way, and centrifuged through gradients containing EDTA and reduced amounts of MgCl2, show a loss of polyribosomes and a large increase in the number of free 40S and 60S subunits (Fig. 2, compare A and B). Concomitantly, there is a shift in the sedimentation pattern of Upf1p from polyribosomes to the lighter fractions of the gradient (Fig. 2, compare panels A and B). These results suggest, but do not prove, that Upf1p is associated with the translation apparatus. EDTA treatment of an extract may also affect other

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activities or structures that require the presence of Mg21. An alternative method by which to disrupt polyribosomes is to treat extracts with limiting concentrations of a ribonuclease. Digestion with micrococcal nuclease cleaves mRNA regions between ribosomes, producing predominantly mRNA fragments bound to only one ribosome. This treatment shifts the dis-

FIG. 1. Factors required for mRNA decay fractionate with polyribosomes on sucrose gradients. Extracts prepared from cells of ySWP154 pRS314/UPF1, yHF1300 YEplac112/ADH1p-HANMD2, yHF1200 YEplac112/ADH1p-HA-UPF3, and yRP582 YEp420/XRN1 were fractionated on 15–50% sucrose gradients and analyzed by Western blotting. Top: A representative OD254 profile of a standard 11-ml sucrose gradient, with sedimentation from right to left. The 80S, 60S, and 40S peaks are indicated by arrows. Bottom: Western blots specific for the indicated proteins. For all but the Upf3p gradient, the entire sample was electrophoresed for the P (pellet) and fractions 1– 8, but only one-fifth of fractions 9 –13 was loaded. For the Upf3p gradient, the entire sample was electrophoresed for the P and fractions 1–9, but only one-fifth of fractions 9 –11 was loaded.

tribution of Upf1p to the top of the gradient (Fig. 2, compare A with C), again suggesting that Upf1p is associated with the translation apparatus. However, from this experiment alone, the only firm conclusion that can be drawn is that Upf1p is associated with a large complex that contains RNA. A more specific test for the significance of a factor’s association with polyribosomes is to assess such association in strains where translation initiation is inhibited. One example of this approach is to use a strain harboring prt1-1, a temperature-sensitive allele of a gene encoding a subunit of initiation factor eIF3 (30). Extracts prepared from the mutant cells grown at the restrictive temperature show that translation initiation is almost completely inhibited; i.e., they contain a large number of ribosomes, but almost no polyribosomes (relative to extracts from cells grown at the permissive temperature; compare Figs. 3A and 3B). In parallel with the inhibition of initiation in prt1-1 cells, the sedimentation of Upf1p again shifts to the lightest fraction of the gradient (Fig. 3, compare A and B). The lack of an increase in Upf1p association with the monoribosomes suggests that the protein associates with the translation apparatus after 60S joining. If the factors required for mRNA decay are present during translation, then they may be tightly associated with the ribosomes. The stringency of this association can be assessed by increasing the salt concentration of both the extract and the buffer in the sucrose gradient (31–33). Ribosomes actively undergoing translation are stable under these conditions while those that are not dissociate into 40S and 60S subunits (Fig. 2, compare A and D). Even with the inclusion of high salt, Upf1p remains tightly bound to small polyribosomes, i.e., those mRNAs that predominantly contain one to three ribosomes (Fig. 2, compare A and D). The high affinity of Upf1p for only small polyribosomes suggests that the strength of its interaction with ribosomes decreases as elongation progresses. Interestingly, such an “early” versus “late” transition is also observed for the destabilzing effects of nonsense codons (10, 11). The polyribosomal association of factors required for mRNA decay suggests that such decay is initiated while the mRNA is still being translated. Thus, it might be expected that intermediates in the decay process would also copurify with the translation apparatus. To test this possibility, we analyzed a PGK1 transcript that contains a poly(G) tract in its 39 untranslated region (UTR).

FIG. 2. Using EDTA disruption, micrococcal nuclease digestion, and high-salt treatment to determine the specificity of Upf1p association with polyribosomes. Extracts prepared from SJ 21R cells were fractionated on 7– 47% sucrose gradients and analyzed by Western blotting. (A) No additional treatment; (B) addition of EDTA; (C) micrococcal nuclease digestion; (D) inclusion of high salt. Top: Relative OD254 profile of 34-ml sucrose gradients, with sedimentation from right to left. The 80S, 60S, and 40S peaks are indicated by arrows. Bottom: Western blots with anti-Upf1p rabbit polyclonal antibody. Fractions 1, 17, and 18 were not included in the Western blot analyses.

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The poly(G) tract blocks 59 3 39 progression by the Xrn1p exonuclease and leads to the accumulation of a small RNA fragment resulting from 39 deadenylation and digestion of the 59 end up to the poly(G) tract (4, 5). The association of this decay intermediate with polyribosomes was assessed by fractionating sucrose gradients and analyzing the samples by Northern blotting. These experiments showed that almost all of the full-length transcript, PGKpG, was present in the polyribosomal fractions of the gradient (Figs. 4A and 4B). A small portion of the decay intermediate, pG, was also present in the polyribosomal fractions, but a much larger fraction sedimented in the lighter regions of the gradient (Figs. 4A and 4C). This suggests that the decay of an mRNA is initiated while the mRNA is still being translated and that partially digested mRNA subsequently dissociates from the translation apparatus.

Materials Equipment Gradient mixer Peristaltic pump (Rainin Rabbit) UV flow cell (Pharmacia UV2) Chart recorder (Fischer Record-All Series 5000) Fraction collector (Gilson FC-80K) Other equipment standard for a molecular biology laboratory Chemicals Cycloheximide (Sigma, C-7698) Dithiothreitol (DTT, Sigma, D-0632) Glass beads (Sigma, G-9268) Heparin (Sigma, H-3393) Phenylmethylsulfonyl fluoride (PMSF, Sigma, P-7626) Protease inhibitors (Boehringer-Mannheim) Sucrose (Sigma, S-7903)

FIG. 3. Using prt1-1 mutant strains to determine Upf1p association with polyribosomes. Extracts from prt1-1 strain TP11B-4-1 grown at the permissive temperature (A; 23°C) or shifted to the restrictive temperature (B; 37°C) for 30 min were fractionated on 7– 47% sucrose gradients and analyzed by Western blotting. Top: Relative OD254 profile of 34-ml sucrose gradients, with sedimentation from right to left. The 80S, 60S, and 40S peaks are indicated by arrows. Bottom: Western blots with anti-Upf1p polyclonal antibody. Fractions 1, 17, and 18 were not included in the Western blot analyses.

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Buffers and Solutions Protease inhibitor cocktail (10003): 0.35 mg/ml bestatin, 0.5 mg/ml leupeptin, and 0.4 mg/ml pepstatin prepared in 100% methanol and stored at 220°C. PMSF stock (0.1 M, 1003): 5 g PMSF in 287 ml 100% 2-propanol, stored at room temperature. Acid-washed glass beads: Soak glass beads in concentrated nitric acid for 1 h, wash several times with water until pH is neutral, and bake until dry in a 200°C oven. Lysis buffer: 10 mM Tris–HCl (pH 7.4 at 20°C), 100 mM NaCl, 30 mM MgCl2, 200 mg/ml heparin.

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Add fresh, 50 mg/ml cycloheximide (if desired), 1 mM DTT, 1 mM PMSF, and 13 protease inhibitors. Chill to 4°C prior to use. Gradient buffers: 50 mM Tris–HCl (pH 7.0 at 20°C), 50 mM NH4Cl, 12 mM MgCl2, and appropriate amount of sucrose. Add fresh, 1 mM DTT. For EDTA treated extracts, reduce MgCl2 to 0.5 mM and add 40 mM EDTA (pH 8.0 at 20°C). For high-salt gradients, add 0.7 M NaCl. Chill to 4°C prior to use. Isolation of Polyribosomes Cell Growth The experiments described here used the following yeast strains: (a) ySWP154 (11), yHF1300 (13), yHF1200 (13), and yRP582 (5), transformed with pRS314/UPF1, YEplac112/ADH1p-HA-NMD2, YEplac112/ADH1p-HAUPF3 (F. He and A.J., unpublished experiments), pRDK252 (34), or pRP469 (4) and (b) SJ 21R and TP11B4-1, related strains, respectively containing the PRT1 gene or its prt1-1 allele (35, 36). Cells were grown in rich medium (YP) with 2% glucose or galactose, but minimal medium or alternative carbon sources may be substituted as required for plasmid maintenance, expression of certain alleles, etc. (37). Cells were grown to log phase and harvested when they reached an OD600 of 0.6–1.0. Preparation of Extracts

FIG. 4. mRNA decay intermediates from PGK1pG cosediment with polyribosomes. Extracts prepared from strain yRP582 transformed with pRP469 were fractionated on 15–50% sucrose gradients. (A) Relative OD254 profile of 12-ml sucrose gradients, with sedimentation from right to left. The 80S, 60S, and 40S peaks are indicated by arrows. RNA was isolated from each fraction, electrophoresed on 6% acrylamide, 7 M urea gels, and analyzed by Northern blotting. A single probe for the poly(G) region identified both the full-length PGK1pG mRNA (B) and resulting mRNA decay intermediate, pG (C). Fraction numbers in (A) correspond to the lanes in (B) and (C).

All procedures and buffers were, respectively, performed or maintained at 0 – 4°C and were developed from methods originally described by Baim et al. (38). A 100-ml culture at the appropriate density (see above) provides sufficient lysate for a single sucrose gradient and is used as the standard volume for this protocol. Cultures are centrifuged at 4000g for 10 min and quickly resuspended in 10 ml lysis buffer (with or without cycloheximide). The cell suspension is transferred to a new tube and centrifuged briefly (until the rotor has reached 3000g), the supernatant is discarded, and the cells are resuspended in 1 ml lysis buffer. This suspension is transferred to 15-ml Corex tubes, supplemented with an equal volume of glass beads, and vortexed at top speed eight times, 15 s each, with 45-s pauses on ice. After lysis, the mixture of beads and broken cells is centrifuged at 12,000g for 10 min. The supernatant is withdrawn to a 1.5-ml centrifuge tube, leaving the pellet and beads behind, and centrifuged in a microcentrifuge at top speed for 10 min. The supernatant (approximately 0.5 ml) is removed and the amount of RNA present is quantitated by reading the value

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of a diluted sample at OD260 in a spectrophotometer. The yield is approximately 20 OD260 units/100 ml cell culture. Variations on this standard protocol allow for assessment of the specificity of factor association. This is achieved by using different conditions to disrupt the integrity of the polyribosomes. To disrupt polyribosomes by chelating Mg21 ions, add EDTA to a final concentration of 40 mM to the extract and centrifuge through sucrose gradients containing EDTA and reduced amounts of MgCl2 (see buffers, above). To digest with ribonuclease, incubate extracts with micrococcal nuclease (S7 nuclease; 500 U/OD260 of extract) and 1 mM CaCl2 for 30 min at 4°C. Stop the reaction by adding EGTA to 4 mM. To disrupt associated ribosomes that are not actively involved in translation, adjust the extract to a final concentration of 0.7 M NaCl and centrifuge through sucrose gradients containing the same concentration of salt. To inhibit translation initiation in cells harboring a prt1-1 allele, grow cultures at 23°C, centrifuge cells for 5 min at 4000g, and resuspend in medium prewarmed to 37°C. Incubate the cells, with shaking, at 37°C for 30 min, and harvest and prepare extracts as usual. Sucrose Gradients Well in advance of extract preparation, use a gradient mixer to pour 7– 47% sucrose gradients in either 12- or 36-ml ultracentrifuge tubes (11 or 34 ml of total sucrose solution, respectively). Gradients of other sizes can be mixed for other applications (e.g., 5–25% sucrose, to assay only monoribosomes, 60S, and 40S subunits). Pipet the high-concentration sucrose (one-half of the total volume) into the mixing chamber and an equal amount of low-concentration sucrose into the other. Be careful to prime the bridge between the chambers with high-concentration sucrose, avoiding an air bubble that would disrupt the mixing of the gradient. Start the mixer, and with a peristaltic pump set at top speed, pour gradients into centrifuge tubes. Store gradients at 4°C until use. Gently layer equal amounts of extract (20 OD260 units), in equal volumes, onto the gradients. For the smaller gradients, centrifuge in a swinging bucket rotor (e.g., Beckman SW41) at 215,000g for 2 h and 45 min. For the larger gradients, centrifuge in a swinging bucket rotor (e.g., Beckman SW28) at 120,000g for 5 h. Harvest each gradient by gently inserting a capillary to just below the taper at the bottom of the tube (or, alternatively, by using a tube

piercer to puncture the bottom of the tube), and pump the gradient through an UV monitor (OD254 filter) with a peristaltic pump. A constant output from the flow cell can be displayed by the attachment of a chart recorder. Collect samples, 0.5–1 or 1–2 ml for the small or large gradients, respectively, using a fraction collector. This basic setup can be assembled with equipment in most laboratories; however, an integrated system can be purchased from Isco, Inc., Lincoln, Nebraska. Analysis of the Gradient Fractions Detection of Specific Proteins To analyze the gradient fractions for the presence of specific proteins, samples are prepared for electrophoresis. The amount of sample required to detect individual factors is dependent on their abundance. For most factors, at least half or the entire sample is electrophoresed. Since the presence of high concentrations of sucrose is detrimental to standard SDS–polyacrylamide gel electrophoresis, it must be removed prior to analysis of the samples. Precipitate the protein from the sample by adding 125 mg/ml sodium deoxycholate and 6% trichloroacetic acid (TCA) (39). Centrifuge in a microcentrifuge at top speed for 10 min and wash the pellet with cold 80% acetone (220°C). Resuspend the pellet in approximately 50 ml of 13 protein gel sample buffer. If the protein pellets are large, TCA may remain trapped and additional Tris–HCl, pH 8.0, should be added to neutralize the sample to prevent acid hydrolysis of the protein. SDS–polyacrylamide gel electrophoresis is performed as described by Laemmli (40) and gels are electroblotted to Immobilon-P (Millipore) using conditions recommended by the manufacturer. Binding conditions for antibodies are as described by Harlow and Lane (41) and detection of binding to specific proteins uses enhanced chemiluminescence (ECL) kits from Amersham Corporation. Blots can be stripped and reprobed according to instructions from the membrane manufacturer. To generate antibodies specific for Upf1p, peptides corresponding to amino acids 925–940 and 954 –970 were synthesized (by the Peptide Synthesis Core Facility at the University of Massachusetts Medical School), coupled to keyhole limpet hemocyanin, and repeatedly injected into New Zealand White rabbits (41). Sera were purified by affinity chromatography with the same synthetic peptides coupled to Sepharose beads (41).

ASSESSMENT OF POLYRIBOSOME ASSOCIATION

RNA Analysis To analyze the gradient fractions for the presence of specific mRNAs, protein and sucrose must be removed from the sample. Add an equal volume of phenol (buffered with Tris to pH 7.6) to each sample. Vortex vigorously and centrifuge in a microcentrifuge for 10 min. Remove the aqueous layer to a new tube, taking care not to transfer any of the protein interface. Note that, due to the high density of some of the sucrose gradient fractions, the aqueous and organic fractions may invert. To prevent this problem, the gradient fractions can be diluted in RNasefree water prior to extraction. Extract the sample with an equal volume of a 1:1 mixture of phenol (buffered with Tris to pH 7.6) and chloroform and centrifuge in a microcentrifuge for 10 min. Remove the aqueous layer and ethanol-precipitate the sample. Centrifuge in a microcentrifuge at top speed for 15 min and wash the pellet with 70% ethanol (220°C). Remove the supernatant and resuspend the pellet in a small volume of denaturing loading dye (42). Heat the samples to 100°C for 3 min before electrophoresing on 6% acrylamide, 7 M urea, 13 TBE gels (42). Transfer the RNA to a membrane by electroblotting (42) and hybridize the blot with a probe specific for the RNA of interest (42).

CONCLUDING REMARKS Factors Required for mRNA Decay Associate with Polyribosomes A growing body of evidence suggests that the processes of mRNA decay and translation are tightly linked (2). Using sucrose gradient fractionation, we have determined that factors involved in mRNA decay, and the resulting degradative intermediates, associate with the translation apparatus. This association may well be transient since only a portion of the decay intermediate and factors assayed cosedimented with polyribosomes. Several methods were used to affirm the specificity of the observed interactions for one of the factors, Upf1p. These assays demonstrated that the sedimentation of Upf1p is altered when polyribosomes are disrupted by treatment with EDTA, digestion with micrococcal nuclease, or shifting of cells containing a prt1-1 allele to 37°C. The same experiments indicated Upf1p association with polyribosomes is not likely to be explained by a simple interaction with one ribosomal subunit: although Upf1p is present in small polyribosomes that result from high-salt treatment of

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lysates, it does not appear to preferentially associate with either the 40S or 60S ribosomal subunits or with 80S monomers that result from micrococcal nuclease digestion of polyribosomes or inhibition of translation initiation in prt1-1 cells. These observations are consistent with other studies that have used similar assays (18, 25). Previous studies have demonstrated that the association of Upf1p with polyribosomes is independent of the presence of Upf3p, but may be slightly altered in the absence of Nmd2p (25). Interestingly, Atkin et al. have observed that the amount of Nmd2p fractionating with polyribosomes increased in a upf1 strain, but was lost from the polyribosomes in a upf3 strain (25). Together, these results are consistent with the idea that these factors function in a linear pathway, dependent on interaction with each other, to regulate translation termination (14, 20, 25). If a determination of mRNA competence for translation is considered at termination (for nonsensecontaining mRNAs) or during initiation, with an assessment of poly(A) tail length (for most other mRNAs), it is reasonable to hypothesize that mRNA decay is triggered while the mRNA is still associated with the translation apparatus. This is almost certainly the case for nonsense-containing mRNAs since their association with the translation apparatus is a prerequisite for their decay (24). Similarly, the polysomal association of a factor required for the decay of most mRNAs (Xrn1p), as well as the identification of a polyribosome-associated mRNA decay intermediate, suggests that decay of general mRNAs also initiates while they are still being translated. The fact that such decay intermediates remain associated with polyribosomes even in the absence of a coding region indicates that a cellular degradation complex may be capable of processing more than one mRNA at a given time. Liabilities of the Assays We have described several methods to affirm factor association with polyribosomes. Each method independently suffers from the indirect nature of the assay; e.g., treatment of extracts with EDTA may affect other activities or structures that require the presence of Mg21 and digestion with ribonuclease may destroy other RNA-containing complexes. Probably the most reliable method to disrupt polyribosomes is to inhibit translation initiation with a temperature-sensitive translation factor mutant. However, even this method is not without its liabilities since inhibiting translation initiation could have indirect consequences, such as disruption of an mRNA degradation complex due to

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the cellular stresses caused by the lack of protein production. However, when taken together, these tests provide a compelling argument to support factor association with polyribosomes. Alternative Treatments Many variations of the specificity tests we have applied could be used. As an alternative treatment to micrococcal nuclease digestion of mRNA, other laboratories have used RNase A (25). We consider micrococcal nuclease to be the preferred reagent since it is possible to regulate its activity by providing or removing Ca21 ions. Instead of treating extracts with 0.7 M NaCl, other laboratories have used 0.8 M KCl. We chose NaCl because it appears to be much less detrimental to in vitro translation assays than other salts. Drugs (e.g. adrenochromre, showdomycin, and edeine A1; 43) may also be used to prevent translation initiation. However, they have the potential disadvantages of altering other cellular processes and being difficult to obtain. The prt1-1 temperature-sensitive allele used to inhibit translation initiation has a very potent effect on initiation, reducing translation to 3–5% of wild-type levels (E. M. Welch and A.J., manuscript in preparation). Other temperature-sensitive mutants might also be used, with the caveat that other mutations in PRT1 do not inhibit translation initiation to the same extent as the prt1-1 allele and do not have the same effects as that allele for nonsense-mediated mRNA decay (E. M. Welch and A.J., manuscript in preparation).

ACKNOWLEDGMENTS This work was supported by a grant to A.J. from the National Institutes of Health (GM 27757). We thank He Feng, Arlen Johnson, and Roy Parker for plasmids; He Feng, Jim Hopper, Gerry Johnson, and Roy Parker for strains; Agneta Brown, Duane Jenness, Arlen Johnson, and Jonathan Warner for antibodies; Jonathan Belk for technical help; and members of our laboratory for comments on the manuscript.

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