Cap-binding 1-mediated and eukaryotic translation initiation factor 4E-mediated pioneer rounds of translation in yeast

Qinshan Gao, Biswadip Das, Fred Sherman, and Lynne E. Maquat*

Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, University of Rochester, Rochester, NY 14642

Contributed by Fred Sherman, January 26, 2005 Nonsense-mediated mRNA decay (NMD) in mammalian cells is initiation complex, it is structurally and functionally distinct (1, .(restricted to newly synthesized mRNA that is bound at the 5؅ cap 2, 4, 5 by the major nuclear cap-binding complex and at splicing-gener- To date, comparable studies of Saccharomyces cerevisiae that ated exon–exon junctions by exon junction complexes. This mes- examine the nature of messenger ribonucleoprotein (mRNP) senger ribonucleoprotein has been called the pioneer translation that is targeted for NMD have not been performed. In this study, initiation complex and, accordingly, NMD occurs as a consequence we demonstrate that NMD in S. cerevisiae targets mRNAs that of nonsense codon recognition during a pioneer round of trans- are bound by the primarily nuclear cap-binding complex lation. Here, we characterize the nature of messenger ribonucle- (Cbc)1p, which is orthologous to mammalian CBP80, and oprotein that is targeted for NMD in Saccharomyces cerevisiae. mRNAs that are bound by the primarily cytoplasmic eIF4E. Data indicate that NMD targets both cap-binding complex (Cbc)1p- Because the ratio of intron-containing pre-mRNA to the cor- and eukaryotic translation initiation factor (eIF)4E-bound mRNAs, responding spliced mRNA product is similar before and after unlike in mammalian cells, where NMD does not detectably target immunopurification (IP) with anti-eIF4E, but at least 4-fold eIF4E-bound mRNA. First, intron-containing pre-mRNAs in yeast larger after IP with anti-Cbc1p, we conclude that eIF4E replaces are detectably bound by either Cbc1p, or, unlike in mammalian Cbc1p at the caps of most transcripts but, alternatively, may bind cells, eIF4E, indicating that mRNAs can be derived from either to the caps of some transcripts without prior Cbc1p binding. This Cbc1p- or eIF4E-bound pre-mRNAs. Second, the ratio of nonsense- replacement of Cbc1p by eIF4E apparently occurs despite the containing Cbc1p-bound mRNA to nonsense-free Cbc1p-bound fact that Cbc1p facilitates pre-mRNA splicing (6) and, possibly, mRNA, which was < 0.4 for those mRNAs tested here, is essentially mRNA export (7, 8). Consistent with the ability of eIF4E to identical to the ratio of the corresponding nonsense-containing productively bind to newly synthesized caps without prior Cbc1p eIF4E-bound mRNA to nonsense-free eIF4E-bound mRNA, and both binding, and in keeping with previous data indicating that cells ratios increase in cells treated with the translational inhibitor lacking Cbc1p are viable and able to support NMD (8–11), we cycloheximide (CHX). These data, together with data presented find that NMD targets eIF4E-bound mRNA in a cbc1-⌬ strain. here and elsewhere showing that Cbc1p-bound transcripts are These data and other data indicate that mRNAs in yeast, like precursors to eIF4E-bound transcripts, demonstrate that Cbc1p- mRNAs in mammalian cells, undergo a Cbc1p-mediated pioneer bound mRNA is targeted for NMD. In support of the idea that round of translation that is inhibited by CHX, and that nonsense eIF4E-bound mRNA is also targeted for NMD, eIF4E-bound mRNA is codon recognition during this round of translation leads to targeted for NMD in strains that lack Cbc1p. These results suggest NMD, provided the Upf NMD factors are present. Additionally, that both Cbc1p- and eIF4E-mediated pioneer rounds of translation these data indicate that mRNAs in yeast also undergo NMD occur in yeast. when bound by eIF4E, unlike mRNAs in mammalian cells.

messenger ribonucleoprotein ͉ nonsense-mediated mRNA decay ͉ Materials and Methods premature termination codon Yeast Strains, Media, and Yeast Genetics. The genotypes of S. cerevisiae strains used in this study are listed in Table 1. Standard tudies of mammalian cells indicate that nonsense-mediated yeast extract͞peptone͞dextrose and omission media were used SmRNA decay (NMD) targets newly synthesized mRNA as a for yeast propagation and testing (12). The UPF3, UPF1, and consequence of nonsense codon recognition during what has UPF2 were sequentially disrupted in the strain B-10529 by been called a pioneer round of translation (1). For spliced using PCR-generated DNA fragments that harbored the appro- mRNAs, the pioneer translation initiation complex is charac- priate fragment and the selective blaster hisG-URA3-hisG terized by (i) the mostly nuclear but shuttling cap-binding (13), thus generating B-15334 (Table 1). heterodimer that consists of cap-binding protein (CBP)80 and CBP20; (ii) the exon junction complex of that is Plasmids. The plasmids used in this study are listed in Table 2. The deposited as a consequence of pre-mRNA splicing Ϸ 20–24 nt PGK1 gene lacking a premature termination codon (PTC) is upstream of exon–exon junctions, and includes NMD factors denoted as PGK1 WT, even though it is not a WT gene. up-frameshift (Upf)2 and Upf3 or Upf3X (also called Upf3a and Upf3b, respectively); and (iii) poly(A)-binding protein (PABP) IPs. Yeast cells were grown in 600 ml of yeast extract͞peptone͞ Ϸ ϫ 7 N1 [previously designated PABP2 (1, 2)] and PABPC [previ- dextrose or SC-Ura medium to an OD600 of 1.5 ( 3 10 cells per ously designated PABP (ref. 2 and F. Lejeune and L.E.M., unpublished data)]. By the time eukaryotic initiation factor (eIF) 4E, which is the major cytoplasmic cap-binding protein, replaces Abbreviations: NMD, nonsense-mediated mRNA decay; mRNP, messenger ribonucleopro- ͞ tein; Upf, up-frameshift; CBP, cap-binding protein; PABP, poly(A)-binding protein; Cbc, the CBP80 20 heterodimer at the mRNA cap, the exon–junction cap-binding complex; eIF, eukaryotic translation initiation factor; IP, immunopurification; complex, and associated Upf NMD factors have been removed NRS, normal rabbit serum; PTC, premature termination codon; CHX, cycloheximide. so that the resulting steady-state mRNA is immune to NMD *To whom correspondence should be addressed. E-mail: lynne࿝maquat@urmc. (1–4). Thus, whereas the pioneer translation initiation complex rochester.edu. has components in common to the steady-state translation © 2005 by The National Academy of Sciences of the USA

4258–4263 ͉ PNAS ͉ March 22, 2005 ͉ vol. 102 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500684102 Downloaded by guest on September 26, 2021 Table 1. Yeast strains Strain no. Genotype Source

B-10529 MAT␣ cyc1-512 lys2-187 leu2-1 ura3-52 his4-166 met8-1 (Normal A) —* B-15315 MAT␣ cyc1-512 lys2-187 leu2-1 ura3-52 his4-166 met8-1 upf3::hisG URA3 hisG B-10529 B-15316 MAT␣ cyc1-512 lys2-187 leu2-1 ura3-52 his4-166 me 8-1 upf3::hisG B-15315 B-15317 MAT␣ cyc1-512 lys2-187 leu2-1 ura3-52 his4-166 met8-1 upf3::hisG ::hisG URA3 hisG B-15316 B-15318 MAT␣ cyc1-512 lys2-187 leu2-1 ura3-52 his4-166 met8-1 upf3::hisG upf1::hisG B-15317 B-15333 MAT␣ cyc1-512 lys2-187 leu2-1 ura3-52 his4-166 met8-1 upf3::hisG upf1::hisG ::hisG URA3 hisG B-15318 B-15334 MAT␣ cyc1-512 lys2-187 leu2-1 ura3-52 his4-166 met8-1 upf3::hisG upf1::hisG upf2::hisG B-15333 B-11621 MATa cyc1-512 lys2-187 his4-166 —† B-11622 MATa cyc1-512 lys2-187 his4-166 leu2-1 met8-1 —† B-9037 MATa cyc1-512 trp2-1 ura3-52 (Normal B) Ref. 11 B-11558 MATa cyc1-512 trp2-1 ura3-52 cbc1::hisG Ref. 11

*B-10529 is a segregant from the diploid strain D-1135. †B-11621 and B-11622 are segregants from the diploid strain B-11768.

ml). Cells were pelleted at 5,000 ϫ g for 5 min, washed with water, using the primers 5Ј-GTCTCTACTGGTGGTGGTG-3Ј (sense) and resuspended in 2 ml of lysis buffer (50 mM Tris⅐HCl, pH and 5Ј-GGGGAAAGAGAAAAGAAAAAAATTGATCTAT- 7.4͞150 mM NaCl͞0.05% Tween 20͞5 mM MgCl2͞1 mM PMSF͞1 CGATAGT-3Ј (antisense). MET8 mRNA that was either WT or mM benzamidine͞10 mM NaF͞10 mM ␤-glycerophosphate). Cells were broken by two passages through a French press at 750 psi, and then centrifuged at 10,000 ϫ g for 10 min. For each IP, 1 ml of supernatant (that corresponded to Ϸ7.5 ϫ 109 cells) was precleared with 50 ␮l of protein A-conjugated agarose (Roche Diagnostics) by rotation for 30 min at 4°C. IP was then performed as described (1). However, 2 mg of bacterial tRNA (Sigma) was used to saturate the beads before IP, and the beads were washed 10 times before elution.

Western Blotting. Western blotting was performed (1) by using the following antibodies: anti-Cbc1p (15) (1:100,000 dilution) or anti-eIF4E (16) (1:1,000 dilution).

RT-PCR. Immunopurified transcripts or transcripts before IP were analyzed by RT-PCR (17). ACT1 pre-mRNA was amplified by using the primers 5Ј-GTCTCATGTACTAACATCGATTGC-3Ј (sense) and 5Ј-CCCAGTTGGTGACAATACCGTG-3Ј (anti- sense), and ACT1 mRNA was amplified, using primer 5Ј- TTTACTGAATTAACAATGGATTCTGAGG-3Ј (sense) and the same antisense primer as used for pre-mRNA. TUB3 pre- mRNA was amplified by using the primers 5Ј-TTTGTGTCT-

TCTTCTTCGG-3Ј (sense) and 5Ј-ATCGATAACATT- BIOCHEMISTRY GGGCTCT-3Ј (antisense), and TUB3 mRNA was amplified, using primer 5Ј-GTCATTAGTATTAATGTTGGT-3Ј (sense) and the same antisense primer as used for pre-mRNA. KIN28 pre-mRNA was amplified by using the primers 5Ј-GTACA- CAAAGGTAGTGGGGG-3Ј (sense) and 5Ј-CTTAACT- TCACGGATAGCTGAC-3Ј (antisense), and KIN28 mRNA was amplified, using primer 5Ј-GAATATGGAGTACACAAAG- GAAAAG-3Ј (sense) and the same antisense primer as used for Fig. 1. The ratio of pre-mRNA to its product mRNA is higher in IPs that used pre-mRNA. CYH2 pre-mRNA was amplified by using the prim- anti-Cbc1p instead of anti-eIF4E. (A) Anti-Cbc1p and anti-eIF4E specifically im- ers 5Ј-GTATCAAATGGTTGTAGAGAGCGC-3Ј (sense) and munopurify Cbc1p and eIF4E, respectively. B-10529 (Normal A) cells were cultured 5Ј-TGTGGAAGTATCTCATACCAACC-3Ј (antisense), and in yeast extract͞peptone͞dextrose medium. Lysates were then generated and CYH2 mRNA was amplified, using primer 5Ј-CAGAGGT- immunopurified by using anti-Cbc1p or anti-eIF4E. The specificity of each IP was CACGTCTCAGCC-3Ј (sense) and the same antisense primer as controlled for by using rabbit IgG or NRS, respectively. Samples were analyzed by Western blotting. IP efficiencies were 20% for Cbc1p and 10% for eIF4E. The five used for pre-mRNA. URA3 mRNA was amplified by using the Ϫ Ј Ј leftmost lanes represent twofold dilutions of protein before IP ( ) and demon- primers 5 -GTGCTTCATTGGATGTTCGTACC-3 (sense) strate that the conditions used for Western blotting were semiquantitative. (B) and 5Ј-CCACCACACCGTGTGCATTC-3Ј (antisense). PGK1 Both intron-containing pre-mRNAs and spliced mRNAs immunopurify with mRNA that was either WT or PTC-containing was amplified by Cbc1p and eIF4E, although to differing extents. RT-PCR was performed to detect the specified transcripts. PCR was initiated by using three amplification cycles and primers for the specified pre-mRNA and continued, using 19 cycles after adding Table 2. Plasmids used in this study primers for the corresponding mRNA, except for both KIN28 pre-mRNA and KIN28 mRNA, which were amplified using 22 cycles. The numbers below each lane Plasmid no. Yeast genes Ref. designate the ratio of pre-mRNA to mRNA after subtracting the amount present pAB2992 [pRIP1PGK(ϪAU)] URA3 PGK1 (WT) 14 in the appropriate control IP. The five leftmost lanes represent twofold dilutions Ϫ pAB2996 [pRIPPGKH2(3)⌬4] URA3 PGK1 (PTC) 14 of RNA before IP ( ) and demonstrate that the conditions used for RT-PCR were semiquantitative.

Gao et al. PNAS ͉ March 22, 2005 ͉ vol. 102 ͉ no. 12 ͉ 4259 Downloaded by guest on September 26, 2021 Fig. 3. Cbc1p-bound MET8 mRNA that harbors a PTC (met8-1) is targeted for NMD. Methods are the same as in Fig. 2B, except that extracts from strains B-11621 (MET8), which harbored a WT MET8 gene, and B-11622 (met8–1), which harbored a PTC-containing MET8 gene, were analyzed. (Upper) West- ern blotting demonstrated that IP efficiencies were 10–20% for Cbc1p and 5–10% for eIF4E. The three leftmost lanes represent twofold dilutions of protein before IP (Ϫ) and demonstrate that the conditions used for Western blotting were semiquantitative. (Lower) RT-PCR demonstrated that the nor- malized level of Cbc1p-bound MET8 PTC mRNA, when presented as a percent- age of the normalized level of Cbc1p-bound MET8 WT mRNA, was comparable to the normalized level of eIF4E-bound MET8 PTC mRNA, when presented as a percentage of the normalized level of eIF4E-bound MET8 WT mRNA. The five leftmost lanes represent twofold dilutions of RNA before IP (Ϫ) and demon- strate that the conditions used for RT-PCR were semiquantitative. ACT1 mRNA was amplified by using 17 cycles, whereas MET8 mRNAs were amplified, using 22 cycles. Fig. 2. PGK1 mRNA that harbors a PTC is targeted for NMD when bound by Cbc1p. (A) PTC-containing PGK1 mRNA is subject to NMD in a way that requires Upf proteins. Yeast strains B-10529 (Normal A) and B-15334 (upf1-⌬ upf2-⌬ scripts in S. cerevisiae, we compared the relative abundance of upf3-⌬) were transformed with a yeast-centromeric plasmid carrying either a intron-containing pre-mRNA and its product, spliced mRNA, in WT or PTC-containing PGK1 gene and the URA3-selective marker. RNA was IPs by using antibody to each cap-binding protein. Strain purified, and RT-PCR was performed to quantitate the levels of PGK1 mRNA B-10529 (Normal A) was grown, and extracts were prepared with after normalization to the level of URA3 mRNA. The normalized level of PGK1 and without IP by using (i) anti-Cbc1p, (ii) anti-eIF4E, (iii) WT mRNA in each strain was defined as 100%. The five leftmost lanes purified rabbit IgG, which controlled for nonspecific IP using represent twofold dilutions of RNA and demonstrate that the conditions used anti-Cbc1p, or (iv) normal rabbit serum (NRS), which controlled for RT-PCR were semiquantitative. Each mRNA was amplified by using 19 for nonspecific IP, using anti-eIF4E. Western blotting demon- cycles. (B) The levels of Cbc1p- and eIF4E-bound PGK1 PTC mRNAs were strated that anti-Cbc1p specifically immunopurified Cbc1p, but reduced to the same percentage of Cbc1p- and eIF4E-bound PGK1 WT mRNAs, not eIF4E, whereas anti-eIF4E specifically immunopurified respectively. IPs were performed as described in Fig. 1A by using the same eIF4E, but not Cbc1p (Fig. 1A). RT-PCR demonstrated that the B-10529 (Normal A) lysates as described in A.(Upper) Western blotting dem- onstrated that IP efficiencies were 20% for Cbc1p and 10% for eIF4E. The six ratio of pre-mRNA to mRNA in the anti-Cbc1p IP was 15- to leftmost lanes represent twofold dilutions of protein before IP (Ϫ) and dem- 21-fold, 4- to 7-fold, 5- to 6-fold, and 4- to 5-fold higher than in onstrate that the conditions used for Western blotting were semiquantitative. the anti-eIF4E IP, respectively, for ACT1, TUB3, KIN28, and (Lower) RT-PCR demonstrated that the normalized level of Cbc1p-bound CYH2 transcripts (Fig. 1B; data not shown for an independently PGK1 PTC mRNA, when presented as a percentage of the normalized level of performed experiment). Because the amplification efficiency of Cbc1p-bound PGK1 WT mRNA, was comparable to the normalized level of each pre-mRNA is likely to differ from that of the corresponding eIF4E-bound PGK1 PTC mRNA, when presented as a percentage of the nor- mRNA, it was not possible to determine the relative amount of malized level of eIF4E-bound PGK1 WT mRNA. The four leftmost lanes rep- each pre-mRNA and mRNA that was associated with each CBP. resent twofold dilutions of RNA before IP (Ϫ) and demonstrate that the However, we can conclude that some pre-mRNA detectably conditions used for RT-PCR were semiquantitative. PGK1 mRNAs were ampli- associates with eIF4E in yeast, which is in contrast to the fied by using 22 cycles, whereas URA3 mRNA was amplified using 19 cycles. situation in mammalian cells (4). Whether eIF4E binds pre- mRNA instead of Cbc1p, after replacing Cbc1p, or both, remains to be determined (see below). Furthermore, we can conclude PTC-containing was amplified by using primers 5Ј-GAGGAAT- Ј Ј that mRNA preferentially associates with eIF4E because the TAGGCTGCTGGCAC-3 (sense) and 5 -CTGCAAGGAA- ratio of pre-mRNA to mRNA is comparable before or after IP Ј CAGTTCTGTTC-3 (antisense). by using anti-eIF4E. Notably, the amount of Cbc1p-bound pre-mRNA was too small to detectably change the ratio of Results pre-mRNA to mRNA before and after IP with anti-eIF4E. The Ratio of pre-mRNA to Its Product mRNA Is Higher in IPs That Used Anti-Cbc1p Instead of Anti-eIF4E. To gain insight into the precursor- NMD Targets Cbc1p-Bound PGK1 mRNA. Because data indicate that product relationship between Cbc1p- and eIF4E-bound tran- pre-mRNA is bound by either Cbc1p or eIF4E, it may be that

4260 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500684102 Gao et al. Downloaded by guest on September 26, 2021 Fig. 4. eIF4E-bound PGK1 mRNA that harbors a PTC is targeted for NMD in a cbc1-⌬ strain. (A) PGK1 PTC mRNA is targeted for NMD in Normal B and cbc1-⌬ strains to the same extent. Methods are the same as in Fig. 2A, except that B-9037 (Normal B) and B-11558 (cbc1-⌬) strains were analyzed. (B) Analysis of eIF4E-bound transcripts in Normal B and cbc1-⌬ strains. (Upper) Western blotting demonstrated that IP efficiencies were 5–10% for eIF4E. The five leftmost lanes represent twofold dilutions of protein before IP (Ϫ) and demonstrate that the conditions used for Western blotting were semiquantitative. (Lower) The ratio of ACT1 pre-mRNA and mRNA is essentially the same in the two strains. The same applies to the ratio of KIN28 pre-mRNA to mRNA. (C) eIF4E-bound mRNA is targeted for NMD in a cbc1-⌬ strain. IPs, Western blotting, and RT-PCR were as in Fig. 2B except that the anti-Cbc1p IP was omitted.

Cbc1p is replaced by eIF4E before the first round of translation. anti-Cbc1p or anti-eIF4E (Fig. 6, which is published as support-

If so, then a Cbc1p-initiated pioneer round of translation would ing information on the PNAS web site). Furthermore, the ratio BIOCHEMISTRY not occur in yeast. To address this possibility, we determined of Cbc1p-bound PGK1 mRNA that does and does not harbor a whether Cbc1p is replaced by eIF4E before NMD. Strains PTC, and the ratio of eIF4E-bound mRNA that does and does B-10529 (Normal A) and B-15334 (upf1-⌬ upf2-⌬ upf3-⌬) were not harbor a PTC, were increased to essentially the same extent transformed with a centromeric plasmid that carried (i)a as the ratio of total-cell PGK1 mRNA that does and does not URA3-selective marker and (ii)aPGK1 gene that was either WT harbor a PTC in cells treated with the translational inhibitor (pAB2992) or harbored a TAG PTC (pAB2996). Extracts were CHX (Fig. 7, which is published as supporting information on the prepared with and without IP as described above. RT-PCR of PNAS web site). We conclude that a Cbc1p-mediated pioneer samples before IP demonstrated that the level of PGK1 PTC round of translation does occur in S. cerevisiae. However, NMD mRNA was 28% the level of PGK1 WT mRNA in the Normal may also target eIF4E-bound mRNA (see below). A strain and increased to 95% of PGK1 WT mRNA in the upf1-⌬ upf2-⌬ upf3-⌬ strain (Fig. 2A), which is consistent with previous NMD Targets Cbc1p-Bound MET8 mRNA. To determine whether our reports (14). In these analyses and subsequent experiments, the finding that NMD targets Cbc1p-bound PGK1 mRNA applies to level of URA3 mRNA was used to control for variations in another Cbc1p-bound mRNA, we assessed met8–1 mRNA, plasmid copy number and RNA recovery. Western blotting which contains an UAG PTC. Extracts were prepared from revealed that the IPs were specific (Fig. 2B Upper). RT-PCR of strain B-11621 (MET8), which harbored a WT MET8 gene, and samples before and after IP revealed that the level of PGK1 PTC strain B-11622 (met8–1), which harbored a PTC-containing mRNA was 20–23% of PGK1 WT mRNA before or after IP with MET8 gene, with and without IP as described above. Western either anti-Cbc1p or anti-eIF4E (Fig. 2B Lower). Because at blotting revealed that the IPs were specific (Fig. 3 Upper). By least some Cbc1p binds to transcripts before eIF4E, and because using the level of ACT1 mRNA to control for variations in RNA some mRNA is bound by Cbc1p, these data indicate that NMD recovery, RT-PCR analysis of samples before and after IP targets Cbc1p-bound mRNA and, therefore, Cbc1p-bound revealed that the level of met8–1 (MET8 PTC) mRNA was the mRNA must be translated. Consistent with this interpretation, same percentage of the level of MET8 WT mRNA before or the level of PGK1 PTC mRNA in the upf1-⌬ upf2-⌬ upf3-⌬ strain after IP with either anti-Cbc1p or anti-eIF4E (Fig. 3 Lower). was 96–102% of PGK1 WT mRNA before or after IP with either Therefore, Cbc1p-bound MET8 mRNA, and possibly, eIF4E-

Gao et al. PNAS ͉ March 22, 2005 ͉ vol. 102 ͉ no. 12 ͉ 4261 Downloaded by guest on September 26, 2021 bound MET8 mRNA, are targeted for NMD. These data indicate that a Cbc1p-mediated pioneer round of translation is not particular to PGK1 mRNA but applies to at least one other, and possibly, other if not all, mRNAs.

eIF4E-Bound PGK1 mRNA Is Targeted for NMD in a cbc1-⌬ Strain. Given that eIF4E binds to the caps of a fraction of pre-mRNAs, and therefore, the caps of their product mRNAs, and because a PTC reduces the levels of Cbc1p-bound mRNA and eIF4E- bound mRNA to the same percentage of normal, NMD could target not only Cbc1p-bound mRNA but also eIF4E-bound mRNA. To determine whether eIF4E can bind to pre-mRNAs before Cbc1p, and to gain a better understanding of why cbc1-⌬ strains are viable and support NMD (11), we transformed strains B-9037 (Normal B) and B-11558 (cbc1-⌬) with the same plasmid that produced PGK1 WT or PGK1 PTC mRNA as described above. As expected, RT-PCR demonstrated that the level of PGK1 PTC mRNA was 26% the level of PGK1 WT mRNA in the Normal B strain, and this percentage was essentially the same in the cbc1-⌬ strain (Fig. 4A), which is consistent with previous reports (11). Western blotting revealed that the IP that used anti-eIF4E was successful (Fig. 4B Upper). RT-PCR of samples before and after IP revealed that (i) the ratio of eIF4E-bound ACT1 pre-mRNA to eIF4E-bound ACT1 mRNA manifested no detectable difference in the presence or absence of Cbc1p (Fig. 4B Lower; the lack of a detectable difference may reflect the low abundance of pre-mRNA relative to mRNA as measured by using RT-PCR); and similarly, (ii) the ratio of eIF4E-bound KIN28 pre-mRNA to eIF4E-bound KIN28 mRNA manifested Fig. 5. Model for the translation of Cbc1p- and eIF4E-bound mRNAs in S. no detectable difference in the presence or absence of Cbc1p cerevisiae. In the nucleus, a yeast gene (thick bar) is transcribed so as to (Fig. 4B Bottom). Furthermore, in a separately performed IP produce mRNA that is bound at the 5Ј cap by either Cbc1p, which binds as a using the same strains and anti-eIF4E (Fig. 4C Top), the level of heterodimer with Cbc2p, or eIF4E. After mRNA export to the cytoplasm (21), PGK1 PTC mRNA was the same percentage of PGK1 WT both Cbc1p- and eIF4E-bound mRNAs initially undergo a pioneer round of mRNA before or after IP, regardless of whether Cbc1p was translation. Cbc1p-bound mRNA is remodeled to eIF4E-bound mRNA during present (Fig. 4C Middle and Bottom). These data suggest that a or after the pioneer round. Additionally, both Cbc1p- and eIF4E-bound pio- fraction of newly synthesized caps normally binds eIF4E without neer translation initiation complexes undergo other steps of mRNP remodel- ing that include the loss and acquisition of mRNA-binding proteins (22). Data prior binding to Cbc1p. These data also indicate that eIF4E- presented here and elsewhere (see Discussion) indicate that NMD can target bound mRNA can be targeted for NMD in the absence of Cbc1p, either Cbc1p- or eIF4E-bound mRNA during any round of translation provided and most likely, also in the presence of Cbc1p (see Discussion). that an mRNA harbors the appropriate nonsense codon. We conclude that whereas normally a fraction of eIF4E-bound PGK1 mRNA derives from Cbc1p-bound PGK1 mRNA (Fig. 1), eIF4E can bind to PGK1 transcripts without prior binding by mediated pioneer round of translation is not essential for a level Cbc1p. of protein synthesis that supports cell viability or for NMD. Consistent with this conclusion, a pioneer round of translation Discussion can alternatively involve eIF4E-bound mRNA. Whether Cbc1p-bound mRNA is translated so that a Cbc1p- We also demonstrate that RNA metabolism in S. cerevisiae mediated pioneer round of translation occurs in S. cerevisiae has and in mammalian cells manifests significant differences. First, been a matter of debate. Cbc1p stimulates translation 2.5-fold intron-containing pre-mRNAs in yeast can be bound by either when steady-state translation initiation is impaired in extracts Cbc1p or eIF4E (Fig. 1). In contrast, intron-containing pre- prepared from strains harboring a mutated form of eIF4G that mRNAs in mammalian cells are detectably bound only by interacts only weakly with eIF4E and the poly(A)-binding pro- CBP80. Second, NMD in yeast targets both Cbc1p- and eIF4E- tein (18). Moreover, eIF4E antagonizes the interaction of Cbc1p bound mRNA (Figs. 2–4; see model in Fig. 5). In contrast, NMD with eIF4GI (18). Whereas these data indicate that Cbc1p can in mammalian cells detectably targets only CBP80-bound direct translation initiation, subsequent studies led to the oppo- mRNA (1, 2, 4). Nevertheless, neither Cbc1p- nor eIF4E-bound site conclusion. In these studies, growth rate, protein synthesis, mRNAs in yeast were detectably associated with the Upf3p and composition of the transcriptome were not affected in NMD factor under conditions where both types of mRNA were strains expressing a point-mutated eIF4G that no longer inter- detectably associated with other mRNP proteins such as Pab1p acted with Cbc1p (19). However, these studies did not indicate (data not shown). In contrast, CBP80-bound mRNA in mam- that Cbc1p does not support translation but only that Cbc1p is malian cells is detectably bound by the orthologous Upf3 and not essential for translation, as we have previously shown (11), Upf3X proteins (1, 4). and in experiments reported here (Fig. 4). Furthermore, Cbc1p In support of our finding that NMD targets newly synthesized functions in ways other than those that can be attributed to its Cbc1p- or eIF4E-bound mRNA, NMD in yeast has been shown interacting with eIF4G (19), which is consistent with our dem- to take place without significant shortening of the mRNA onstrating a dependence of nuclear mRNA degradation on poly(A) tail (23, 24). Furthermore, the RNA-binding protein Cbc1p (20). In this communication, we provide the first indica- Hrp1p͞Nab4p, which has nuclear roles in transcript processing tion, to our knowledge, that Cbc1p-bound mRNA can be trans- and export, associates with a putative destabilizing element early lated and that normal WT strains of yeast undergo a Cbc1p- in the biogenesis of mini-PGK1 mRNA and reportedly recruits mediated pioneer round of translation. However, a Cbc1p- Upf1p (possibly analogously to how the exon–junction complex

4262 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500684102 Gao et al. Downloaded by guest on September 26, 2021 recruits Upf1) so as to elicit NMD if there is an upstream to an extent that might not be limited to the newly synthesized nonsense codon (25). Nevertheless, other studies of yeast indi- mRNA (28). cate that NMD also targets steady-state mRNA, indicating that Future studies will undoubtedly shed additional light on the mRNP proteins that are required for NMD are not shed during nature of Cbc1p-bound mRNA translation and its significance to translation or are shed but can reassociate. In support of NMD NMD in yeast. targeting steady-state mRNA, nonsense-containing transcripts in cells in which RNA polymerase II had been thermally Note. After completion of this work, Kuperwasser et al. (21) reported inactivated (i) accumulated on polysomes in the presence of that NMD occurs minimally if at all on Cbc2p-bound mRNA (Cbc2p is the yeast ortholog to mammalian CBP20), and they argued against a CHX and (ii) continued to be degraded (i.e., were lost from Cbc1p͞Cbc2p-mediated pioneer round of translation. These authors polysomes) once CHX had been washed away (26). Because used GFP gene constructs that contained or lacked a PTC (PTCϩ or polysomes consist largely of steady-state mRNA, the resumed PTCϪ, respectively), and they performed IPs of cells expressing V5- disappearance of polysome-associated mRNA after the removal tagged Cbc2p by using anti-V5. Comparable amounts PTCϩ and PTCϪ of CHX suggests that NMD targets steady-state mRNA. Also in mRNAs were found after IP by using anti-V5 when normalized to the support of NMD targeting steady-state mRNA, nonsense- level of U2 small nuclear RNA, which normally associates with Cbc2p. ϩ containing transcripts underwent decay in cells in which each of In contrast, the level of PTC mRNA without IP was 40% the level of PTCϪ mRNA. It remains to be seen whether the differences reported the three Upf NMD factors, under the control of a galactose- by us in this paper and by Kuperwasser et al. (21) are because of inducible promoter, was induced after a period of repression differences in the IP procedures or their use of a GFP construct. (27). Because the repression of Upf NMD factors lasted long after most mRNAs had been synthesized and exported from We thank Scott Butler, Francoise Stutz, and Jon Dinman for helpful nuclei, the resumed disappearance of cytoplasmic mRNA after conversations; Scott Butler, Fabrice Lejeune, and Yoon Ki Kim for the induction of these factors suggests that NMD targets steady- comments on the manuscript; Fabrice Lejeune for assistance with the state mRNA. NMD has also been argued to target steady-state IPs; Dirk Go¨rlich (Universita¨t Heidelberg, Heidelberg) for anti-Cbc1p; mRNA in experiments that used programmed Ϫ1 ribosomal John McCarthy (University of Manchester Institute of Science and Technology, Manchester, U.K.) for anti-eIF4E; and Allan Jacobson frameshift signals. When these signals were inserted into a (University of Massachusetts Medical School, Worcester) for centro- reporter mRNA so that ribosomes encountered a downstream meric plasmids carrying a WT or PTC-containing PGK1 gene. This work nonsense codon at low frequencies (i.e., 1–12%), decay appeared was supported by National Institutes of Health Grants R01 DK033938 to occur after the transcriptional shutoff of mRNA production (to L.E.M.) and R01 GM12702 (to F.S.).

1. Ishigaki, Y., Li, X., Serin, G. & Maquat, L. E. (2001) Cell 106, 607–617. 16. Lang, V., Zanchin, N. I. T., Lu¨nsdorf, H., Tuite, M. & McCarthy, J. E. G. (1994) 2. Chiu, S-Y., Lejeune, F., Ranganathan, A. C. & Maquat, L. E. (2004) Genes Dev. J. Biol. Chem. 269, 6117–6123. 18, 745–754. 17. Sun, X., Perlick, H. A., Dietz, H. C. & Maquat, L. E. (1998) Proc. Natl. Acad. 3. Kim, V. N., Kataoka, N. & Dreyfuss, G. (2001) Science 293, 1832–1836. Sci. USA 95, 10009–10014. 4. Lejeune, F., Ishigaki, Y., Li, X. & Maquat, L. E. (2002) EMBO J. 21, 3536–3545. 18. Fortes, P., Inada, T., Preiss, T., Hentze, M. W., Mattaj, I. W. & Sachs, A. B. 5. Lejeune, F., Ranganathan, A. C. & Maquat, L. E. (2004) Nat. Struct. Mol. Biol. (2000) Mol. Cell 6, 191–196. 11, 992–1000. 19. Baron-Benhamou, J., Fortes, P., Inada, T., Preiss, T. & Hentze, M. W. (2003) 6. Lewis, J. D., Go¨rlich, D. & Mattaj, I. W. (1996) Nucleic Acids Res. 24, RNA 9, 654–662. 3332–3336. 20. Das, B., Butler, J. S. & Sherman, F. (2003) Mol. Cell. Biol. 23, 5502–5515. 7. Shen, E. C., Henry, M. F., Weiss, V. H., Valentini, S. R., Silver, P. A. & Lee, 21. Kuperwasser, N., Brogna, S., Dower, K. & Rosbash, M. (2004) RNA 10, M. S. (1998) Genes Dev. 12, 679–691. 1907–1915. 8. Shen, E. C., Stage-Zimmermann, T., Chui, P. & Silver, P.A. (2000) J. Biol. 22. Erkmann, J. A. & Kutay, U. (2004) Exp. Cell Res. 296, 12–20. Chem. 275, 23718–23724. 23. Muhlrad, D. & Parker, R. (1994) Nature 370, 578–581. 9. Uemura, H. & Jigami, Y. (1992) J. Bacteriol. 174, 5526–5532. Cell 113, 10. Fortes, P., Kufel, J., Fornerod, M., Polycarpou-Schwarz, M., Lafontaine, D., 24. Cao, D. & Parker, R. (2003) 533–545. Tollervey, D. & Mattaj, I. W. (1999) Mol. Cell. Biol. 19, 6543–6553. 25. Gonza´lez, C. I., Ruiz-Echevarria, M. J., Vasudevan, S., Henry, M. F. & Peltz,

11. Das, B., Guo, Z., Russo, P., Chartrand, P. & Sherman, F. (2000) Mol. Cell. Biol. S. W. (2000) Mol. Cell 4, 489–499. BIOCHEMISTRY 20, 2827–2838. 26. Zhang, S., Welch, E. M., Hogan, K., Brown, A. H., Peltz, S. W. & Jacobson, 12. Sherman, F. (2002) Methods Enzymol. 350, 3–41. A. (1997) RNA 3, 234–244. 13. Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541–545. 27. Maderazo, A. B., Belk, J. P., He, F. & Jacobson, A. (2003) Mol. Cell. Biol. 23, 14. Peltz, S. W., Brown, A. H. & Jacobson, A. (1993) Genes Dev. 7, 1737–1754. 842–851. 15. Go¨rlich, D., Kraft, R., Kostka, S., Vogel, F., Hartmann, E., Laskey, R. A., 28. Plant, E. P., Wang, P., Jacobs, J. L. & Dinman, J. D. (2004) Nucleic Acids Res. Mattaj, I. W. & Izaurraide, E. (1996) Cell 87, 21–32. 32, 784–790.

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