INTRODUCTION

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Chlamydomonas reinhardtii is a biflagellated green alga capable of rapidly and synchronously regenerating amputated flagella. Complete regeneration requires a massive induction of flagellar protein synthesis, mediated by the accumulation of their mRNAs (23). Following flagellar regeneration, induced flagellar protein mRNAs are rapidly degraded, exhibiting half-lives of 5 to 20 min, thereby effectively returning the cells to their normal program of protein synthesis (see, e.g., references 3, 4, 14, and 31). We have exploited this induction as a model system to study the regulated stability of the tubulin mRNAs. The mRNAs encoding the - and -tubulins (the major flagellar proteins) are synthesized both constitutively and in response to deflagellation. Previous studies have shown that the postinduction half-lives of these mRNAs are accelerated about fourfold relative to their constitutive half-lives, although the transcripts are apparently identical (2). Whether all flagellar protein mRNAs exhibit these dual stability characteristics is not known, because the nontubulin mRNAs are present at only very low levels in nonregenerating cells. We are interested in understanding the nature of the postinduction degradation pathway and whether it differs qualitatively from the degradation process involved in the constitutive turnover of the tubulin mRNAs.

While significant progress is being made in mapping sequence elements that influence mRNA stability, determining exactly what effects those sequences exert on the degradation process has been difficult. Little is known about the pathway(s) by which the great majority of cytoplasmic mRNAs are degraded. One of the major reasons for this poor understanding is the rarity of mRNA degradation intermediates stable enough to accumulate in vivo. There are a few known exceptional cases in which naturally long-lived, discrete degradation intermediates are generated. These rare examples have provided evidence that the initial step(s) in the degradation of some mRNAs is one or more specific endonucleolytic cleavages (9, 10, 28, 32, 33, 35). On the other hand, degradation of the oat phytochrome A mRNA yields an array of intermediates, best explained by a combination of 5'-to-3' and 3'-to-5' degrading activities (17).

Remarkable progress toward defining mRNA decay pathways in yeast has resulted from the finding that insertion of a stretch of guanosine nucleotides into an mRNA yields a "trapped" 3' degradation intermediate spanning from the poly(G) tract to the natural 3' terminus of the mRNA (15). The following evidence indicates that the stable fragment that results is the product of mRNA decapping and impeded 5'-to-3' exonuclease activity (reviewed in reference 11). Decapped, full-length products accumulate in cells that are deficient in Xrn1p activity, the major 5'-to-3' exonuclease in yeast (26). 3' decay fragments fail to accumulate in dcp1 strains, which are deficient in decapping activity (8). The major fragment that accumulates in cells expressing constructs with two poly(G) tracts is the one extending from the first poly(G) to the terminus (26). Analysis of the polyadenylation status of trapped decay intermediates has defined two distinct pathways leading to the onset of decapping and exonuclease digestion: one pathway, likely to be the common pathway for many mRNAs, requires deadenylation to an oligo(A) length (15, 26, 27), while the second pathway does not (25). To date, the only known substrates for the second pathway are aberrant mRNAs that contain premature nonsense codons, although there are likely to be others.

While a role for poly(A) shortening in triggering the degradation of some mRNAs has been suspected for some time (reviewed in references 1 and 34), Parker and colleagues provided the first direct demonstration of this relationship. The extent to which this mechanism occurs in other organisms is not yet clear. There is a convincing compilation of evidence that the onset of degradation of mRNAs encoding c-Fos and other early-response proteins in mammalian cells requires deadenylation (reviewed in references 1 and 12). On the other hand, analysis of the length of the poly(A) sequence on the few naturally long-lived 3' decay intermediates identified in plants and animals (see above) provides no evidence for a poly(A) shortening prerequisite for these mRNAs. Recently, Couttet et al. (13) have published data indicating that deadenylation precedes decapping for at least four mammalian mRNAs. Whether 5'-to-3' digestion follows their decapping and whether this is a major or minor decay pathway for these mRNAs could not be addressed by the methods used in that study.

In this study, we have used the approach developed by Decker and Parker (15) to trap degradation intermediates of 1-tubulin mRNA in Chlamydomonas reinhardtii. These studies indicate that 1-tubulin mRNA is normally subject to a deadenylation-dependent, 5'-to-3' exonucleolytic decay process but that its accelerated postinduction decay occurs via a different pathway. This is the first demonstration that a single transcript can be targeted to different decay pathways under different cellular conditions.

	MATERIALS AND METHODS

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Plasmids. Plasmid ptubHApG was derived from plasmid p853, which contains the entire 1-tubulin gene with 5'- and 3'-flanking genomic sequences and sequences encoding a 12-amino-acid influenza virus hemagglutinin epitope tag (CYPYDVPDYASL) adjusted for Chlamydomonas codon bias (21). p853 was a gift from Joel Rosenbaum (Yale University). To begin constructing ptubHApG, a unique NcoI site was introduced into p853 8 bp downstream of the translational termination codon by site-directed mutagenesis. Mutagenesis was accomplished by the method of Kunkel et al. (22) with the mutagenic oligonucleotide sequence 5'-CCCTGATGCCATCCATGGAGTCTAGTAC-3' (underlining indicates sites of base substitutions) to generate p853/NcoI. Two oligonucleotides composed of poly(G)₃₀ and poly(C)₃₀ tracts containing NcoI recognition sequences on both ends were annealed and subsequently digested with NcoI. This fragment was then ligated into NcoI-linearized p853/NcoI to generate ptubHApG. Plasmid ptubHApG² was constructed by insertion of a second poly(G)₃₀ tract in frame at a BstEII site at codon 366 within the 1-tubulin coding region of plasmid ptubHApG. The poly(G)₃₀ tracts of both plasmids were sequenced to confirm their orientation.

Cell culture, transformation, and screening. Chlamydomonas strains 125M⁺ and nit1-305 were obtained from the Chlamydomonas Genetic Stock Center (Duke University). Strain 5C12G12, which expresses the 1-tubulin gene containing the HA epitope tag (tubHA), was also kindly provided by the Rosenbaum laboratory (21). The cells were cultured in minimal medium and deflagellated by mechanical shearing as previously described (3). Cycloheximide (CX; Sigma Chemical Co., St. Louis, Mo.) was added to cultures at a final concentration of 20 µg/ml. Actinomycin D (Act-D; Sigma Chemical Co.) was used at a final concentration of 160 µg/ml (3). It is unknown why such a high concentration of this inhibitor is required for efficient inhibition of transcription in Chlamydomonas; however, the cells tolerate it well, since they swim vigorously throughout the experiments. Transformation was accomplished by the glass bead vortexing method of Kindle (19), using the Chlamydomonas gene for nitrate reductase as the selectable marker for transformation (20). Prior to transformation, the cell walls were removed by autolysin treatment (16). Transformed colonies were expanded on selective medium (in replicate experiments) and screened for expression of tubHApG or tubHApG² protein by Western analysis. Sodium dodecyl sulfate (SDS)-solubilized cell extracts were prepared by the method of Rochaix et al. (29). For Western blot analysis, protein samples were separated by SDS-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose (Schleicher & Schuell, Keene, N.H.). Efficient transfer was monitored by Ponceau S staining. The blots were blocked with 1% nonfat dry milk in TTBS (0.5 M NaCl, 20 mM Tris-HCl [pH 7.5], 0.1% Tween 20) and probed with a polyclonal antiserum (HA.11; Berkeley Antibody Co., Berkeley, Calif.) against the HA epitope. Secondary antibody coupled to alkaline phosphatase was detected with the Western Blue 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT) chromogenic substrate (Promega, Madison, Wis.).

RNA preparation and Northern blot analysis. For each time point, cells were resuspended in RNA lysis buffer (0.3 M NaCl, 5 mM disodium EDTA, 50 mM Tris-HCl [pH 8.0], 2% SDS) at 2 × 10⁷ cells/ml and quick-frozen. Phenol-chloroform extractions were performed as previously described (2). For Northern blots, total nucleic acid was run on 1.4% formaldehyde-agarose gels and vacuum blotted to Nytran membrane (Schleicher & Schuell) in 4× SSC (1× SSC is 0.15 M NaCl plus 15 mM sodium citrate). Methylene blue staining of the blots was routinely performed to evaluate the relative RNA loading and integrity. The filters were hybridized with ³²P-labeled riboprobes specific for the rbcS2 (ribulose bisphosphate carboxylase small-subunit 2) mRNA and 1-tubulin 3' untranslated region (UTR) as previously described (5). Oligonucleotide probes were 5'-end labeled with ³²P by the method of Sambrook et al. (30). The sequence of the oligonucleotide probe used to detect the influenza virus HA epitope sequence is 5'-GGCGTAGTCGGGCACGTCGTAGGGGTA-3' (21). The HA oligonucleotide probe was hybridized in 5× SSC-5× Denhardt's reagent-0.5% SDS-100 µg of sonicated salmon sperm DNA per ml at 60°C overnight. These blots were washed three times at room temperature in 1× SSC-0.1% SDS and once at 60°C in 0.1× SSC-0.1% SDS for 15 min each. The poly(C)₃₀ oligonucleotide probe was hybridized in 6× SSC-10× Denhardt's reagent-0.1% SDS at 60°C overnight. The blots were washed in 6× SSC-0.1% SDS three times at room temperature and once at 70°C for 20 min each.

High-resolution Northern blot poly(A) length analysis. 3' mRNA fragments were generated by oligonucleotide-directed RNase H cleavage as previously described (4). Briefly, an 1-tubulin-specific oligonucleotide was incubated with total nucleic acid, and Escherichia coli RNase H (Promega) was added to cleave the RNA-DNA hybrids. Oligo(dT) was included in some reactions to completely deadenylate the mRNA fragments. The digests were then run on 6% acrylamide-7 M urea gels in 1× TBE (90 mM Tris-borate, 2 mM disodium EDTA [pH 8.0]) and subsequently electroblotted to Nytran membranes. Radiolabeled RNA size markers, prepared by in vitro transcription, were also included in these gels (24). Blots were hybridized, washed, and autoradiographed as described above.

	RESULTS

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A poly(G)-containing 1-tubulin mRNA exhibits stability characteristics like those of the natural 1-tubulin mRNA. The tubulin mRNAs in Chlamydomonas are degraded with no detectable accumulation of decay intermediates, as is the case for the majority of mRNAs in other organisms. The success of using a poly(G) insert to trap exonucleolytic decay intermediates of yeast mRNAs (15) led us to try this approach in Chlamydomonas. We first stably transformed cells with the ptubHApG construct illustrated in Fig. 1. The plasmid contained the entire 1-tubulin genomic sequence, including both 5'- and 3'-flanking regions needed for proper transcriptional induction and 3'-end formation (21). A tract of 30 guanidylate residues was inserted 8 bp downstream of the termination codon within the 3' UTR. The construct also contains a 12-codon influenza virus HA epitope tag immediately upstream of the stop codon. This inserted tag was used to screen for transformants by Western analysis as well as to provide a unique sequence by which to distinguish transgenic from endogenous 1-tubulin mRNA. The mRNA produced from this construct is referred to as the tubHApG mRNA.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the transcribed regions of the ptubHApG and ptubHApG2 constructs and position of relevant probes used in this study. Plasmid ptubHApG contains the entire 1-tubulin gene including 2 kb of upstream genomic sequence and 1.3 kb of flanking downstream sequence. It has two inserted sequences: a 12-codon influenza virus HA epitope tag immediately upstream of the termination codon and a 30-bp poly(G) tract 8 bp downstream of the termination codon. Above the construct is a restriction map of the indicated region. The ptubHApG2 construct contains a second poly(G)30 tract within the 1-tubulin coding region (in frame at codon 366). The positions of the three probes used in this study are shown below the construct. The probes include an in vitro-transcribed riboprobe complementary to 142 bases within the 1-tubulin 3' untranslated region and two antisense oligonucleotide probes complementary to the poly(G)30 tract and the HA epitope sequence.

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Comparison of expression and stability of tubHApG mRNA and 1-tubulin mRNA. (A to D) Cell cultures of a wild-type strain (wt) and a strain transformed with ptubHApG (csHApG) were deflagellated or treated with Act-D at time zero, and total nucleic acid was extracted at various time points for Northern blot analysis. Values above each lane in autoradiograms indicate minutes after deflagellation (DF) (A and C) or after addition of Act-D (B and D). For Northern blots, nucleic acid samples (4 to 6 µg) were run on 1.4% formaldehyde-agarose gels, transferred to a nylon membrane, and hybridized with 32P-labeled probes hybridized with an 1-tubulin 3' UTR probe (A and B) or with an antisense HA oligonucleotide probe (C and D). Each blot was rehybridized with a probe that detects the constitutively expressed, stable rbcS2 mRNA. (E and F) Plots of Northern blot data shown above each graph, quantified by densitometry.  and , 1-tubulin mRNA in wild-type cells;  and , tubHApG mRNA in transformed cells.

A 3' fragment of the poly(G)-containing 1-tubulin mRNA accumulates in both uninduced and deflagellated cells. Figure 3A and B shows Northern blots of total RNA from uninduced and deflagellated cells expressing the tubHApG mRNA (lanes 3 to 8). The blot shown in Fig. 3A was hybridized with an 1-tubulin 3' UTR riboprobe (which detects both 1-tubulin and tubHApG mRNAs). A low-molecular-weight RNA species was detected at all time points; it accumulated during induction and disappeared as induced transcript levels returned to basal levels. Its presence in uninduced cells is difficult to detect in these Northern blots but is obvious in Fig. 3C. This low-molecular-weight species was absent from both wild-type cells (lane 1) and tubHA-expressing cells (lane 2) 30 min after deflagellation. Based on the estimated size of the fragment (~300 nucleotides) and the results of previous studies with yeast, we hypothesized that this RNA species was a decay intermediate spanning from and including the poly(G) tract to the 3' end of the transcript. To show that it contained at least part of the poly(G) sequence, we hybridized a Northern blot of the same set of RNAs with a poly(C)₃₀ oligonucleotide probe and detected the same-sized fragment (Fig. 3B). The poly(C) probe did not detect RNA of the correct size in wild-type or tubHA-expressing cells (lanes 1 and 2). (The signal in lanes 1 and 2 occurs because this probe sticks to 18S rRNA, which migrates slightly higher than the 1-tubulin and tubHApG mRNAs. This sticking is also evident in Fig. 3C, lanes 4 and 5.) Evidence that the fragment extends to the natural 3' terminus of the mRNA is presented later in this report.

View larger version (66K): [in this window] [in a new window]   FIG. 3.   Accumulation and decay of 3' fragments derived from tubHApG and tubHApG2 mRNAs. Stably transformed cell strains expressing the tubHApG mRNA, the tubHApG2 mRNA, or the tubHA mRNA [no poly(G) insert] and wild-type (wt) cells were deflagellated, and nucleic acid was prepared at the time points indicated. Northern blots were prepared as described in the legend to Fig. 2. FL, full-length transcript; DI, decay intermediate. (A) Northern blot of RNAs from wild-type cells (lane 1) and tubHA-expressing cells (lane 2) 30 min after deflagellation and tubHApG-expressing cells before and after deflagellation (lanes 3 to 8). Numbers above lanes 4 to 8 are minutes after deflagellation (DF); 0, nondeflagellated cells. The blot was probed with the 1-tubulin 3' UTR riboprobe. (B) Northern blot of RNAs from the same cell strains as in panel A but probed with the poly(C) oligonucleotide. (C) Northern blot of RNAs from Act-D-treated, nondeflagellated cells, probed with the poly(C) oligonucleotide. Numbers above each lane are hours in Act-D. (D) Northern blot of RNAs from the cell strains indicated prepared before deflagellation (lanes 1, 3, 5, and 7) or at 30 min after deflagellation (lanes 2, 4, 6, and 8), probed with the poly(C) oligonucleotide.

tubHApG decay intermediates are oligoadenylated in nondeflagellated cells and polyadenylated in deflagellated cells. If the 3' fragments of the tubHApG mRNA represent bona fide intermediates in the decay of this transcript, their accumulation provides the opportunity to address whether deadenylation is required for the onset of degradation. If the onset of decay requires deadenylation, the 3' fragments should bear only short or no poly(A) tails. If deadenylation is not required, the fragments should bear a distribution of poly(A) lengths similar to those of the intact mRNA population. These 3' fragments will be referred to hereafter as decay intermediates to distinguish them from in vitro-generated fragments (discussed below).

View larger version (48K): [in this window] [in a new window]   FIG. 4.   Poly(A) status of tubHApG and tubHApG2 mRNAs and their decay intermediates in nondeflagellated and deflagellated cells. For all of the Northern blots shown, 15-µg nucleic acid samples were run on 6% polyacrylamide gels and transferred electrophoretically to nylon membranes. (A) Northern blot showing tubHApG decay intermediates in nondeflagellated cells (lane 3) and in deflagellated cells (lanes 4 to 8). This blot was hybridized with the 1-tubulin 3' UTR riboprobe. Lanes 1 and 2 contain RNA from wild-type (wt) and tubHA-expressing cells, respectively, isolated 30 min after deflagellation (DF). Lane 9 contains deflagellated cell RNA (30-min sample) deadenylated in vitro by incubation with oligo(dT) and RNase H. Brackets depict the poly(A) length distribution observed for the decay intermediate (DI) (A+, longest length; A, deadenylated). The deadenylated 3' fragment migrates slightly faster than predicted relative to a 333-nucleotide RNA marker (data not shown). (B) Northern blot showing tubHApG2 mRNA decay intermediates in nondeflagellated cells (lane 1) and at 30 min after deflagellation (lane 2). Lane 3 shows the same sample as in lane 2 after in vitro deadenylation as described above. (C) Northern blot showing the poly(A) status of intact tubHApG mRNA in deflagellated cells (lanes 2 to 6) and nondeflagellated (NDF) cells (lane 8). Full-length tubHApG mRNAs were cleaved into 5' and 3' fragments by oligonucleotide-directed RNase H digestion. The blot was probed with 32P-labeled antisense oligonucleotide complementary to the HA sequence tag, which recognizes the in vitro-generated 3' cleavage fragments but not the decay intermediate. Lanes 1 and 7 show RNA samples deadenylated in vitro by including oligo(dT) in the RNase H digestion. (D) Northern blot showing the poly(A) status of the tubHApG decay intermediate (DI) and of in vitro-generated 3' mRNA fragments from full-length mRNAs (1-tub, 1-tubulin and tubHApG mRNAs combined). All RNA samples (except that shown in lane 7) were incubated with an oligonucleotide that targets the full-length mRNAs, but not the decay intermediate, for RNase H cleavage. This blot was probed with the 1-tubulin 3' UTR riboprobe. Lane 1 shows the migration of in vitro-deadenylated fragments of 1-tubulin mRNA from wild-type cells (~400 nucleotides), and lane 7 shows in vitro-deadenylated decay intermediates (~315 nucleotides).

Inhibition of protein synthesis blocks both tubHApG mRNA degradation and formation of the decay intermediate in deflagellated cells. Previous studies have shown that the tubulin and other flagellar protein mRNAs accumulate normally when deflagellation occurs in the presence of CX but that their subsequent rapid degradation is blocked (3, 4). To further confirm that the 3' fragment is an authentic decay intermediate, we asked whether inhibition of protein synthesis altered its accumulation. Figure 5A shows a Northern blot of RNA prepared from cells deflagellated in the absence or presence of CX and hybridized with the poly(C) probe. This blot shows that the decay intermediate accumulates to readily detectable levels in cells deflagellated in the absence of CX (lanes 3 through 8) but not in the presence of CX (lanes 10 through 13). The decay intermediate is clearly present in the predeflagellation time point (lane 9). These cells were incubated in CX for 15 min before deflagellation, indicating that exposure to CX does not, by itself, result in rapid loss of the constitutive decay intermediate. Figure 5B shows a high resolution Northern blot of the same CX-containing samples shown in Fig. 5A, hybridized with the tubulin 3' UTR probe. This image confirms that the 3' decay intermediate does not accumulate in cells deflagellated in the presence of CX. It also shows that incubation in CX did not alter the oligoadenylated state of the constitutive decay intermediate (lane 4). We have observed in multiple experiments that the oligoadenylated decay intermediate present at steady state completely disappears after deflagellation (in the presence or absence of CX) and that its disappearance occurs within 15 min. This could be due to activation of a new degradation mechanism in deflagellated cells or to the normal decay of this fragment in the absence of new accumulation.

View larger version (59K): [in this window] [in a new window]   FIG. 5.   Effect of cycloheximide addition on decay intermediate accumulation. Cells expressing the tubHApG mRNA were deflagellated in the absence or presence of CX (20 µg/ml added 15 min before deflagellation). (A) Autoradiogram of a Northern blot probed with the poly(C) oligonucleotide. Numbers above the lanes are minutes after deflagellation (DF), in the absence (lanes 4 to 8) or presence (lanes 10 to 13) of CX. Lane 9 (0 min) shows RNA from nondeflagellated cells exposed to CX for 15 min. Lanes 1 and 2 show RNA from deflagellated wild-type (wt) and tubHA-expressing cell strains. The same blot was rehybridized with the rbcS2 probe. FL, full-length; DI, degradation intermediate. (B) High-resolution Northern blot analysis of RNA samples from CX-treated cells (lanes 4 to 8), hybridized with the 3'-UTR riboprobe. Lanes 3 and 9 show the migration of the in vitro-deadenylated decay intermediate.

	DISCUSSION

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Evidence for deadenylation-dependent decay of 1-tubulin mRNA in Chlamydomonas. This study demonstrates that a poly(G)₃₀ insert in the 3' UTR of 1-tubulin mRNA of Chlamydomonas leads to the accumulation of a 3' fragment of that mRNA. Experiments comparing fragment sizes before and after oligo(dT)-directed RNase H digestion indicate that the fragment terminates at the normal 3' end in oligo(A) or poly(A). Hybridization of Northern blots with a poly(C) probe shows that the fragment contains at least some of the poly(G) tract, and its size is consistent with the poly(G) tract occurring at the 5' terminus. RNA fragments beginning with the poly(G) tract and terminating with oligo(A) accumulate in yeast cells expressing poly(G)-containing mRNAs (15, 26, 27). In yeast, the fragments almost certainly result from the inability of a 5'-to-3' exonuclease (Xrn1p) to proceed efficiently through the poly(G), since in cells lacking Xrn1p activity, full-length (decapped) mRNAs accumulate while the 3' fragment is absent or much reduced (18, 26). There is strong evidence to support the notion that the XRN1 nuclease is a (or the) major mRNA-degrading enzyme in yeast (reviewed in reference 11), arguing that the observed fragments are trapped intermediates of the normal decay process. The accumulation of poly(G)-containing 3' RNA fragments in Chlamydomonas, in particular the accumulation of only the longer fragment in cells expressing the tubHApG² mRNA, strongly suggests that a 5'-to-3' exonuclease might operate to degrade mRNAs in this organism too.

Evidence for a deadenylation-independent decay pathway for 1-tubulin mRNA in deflagellated cells. Most interestingly, we show that the 3' fragments derived from deflagellation-induced tubHApG and tubHApG² mRNAs carry long poly(A) tails. Thus, the deadenylation prerequisite for the degradation process leading to 3' decay intermediates is bypassed during the induction event. Deflagellation-induced - and -tubulin mRNAs exhibit a three- to fourfold-reduced half-life relative to the same transcripts in nondeflagellated cells (2). Bypassing the deadenylation step could contribute to, or be wholly responsible for, this shortened half-life. A deadenylation-independent mRNA decay pathway has also been described in yeast. The only known substrates of this so-called nonsense-mediated decay pathway are mutant or unspliced mRNAs containing premature nonsense codons; accessing this pathway results in accelerated degradation (25).

View larger version (12K): [in this window] [in a new window]   FIG. 6.   Deadenylation-dependent and -independent pathways for the degradation of 1-tubulin mRNA in Chlamydomonas. Analysis of poly(G)-trapped decay intermediates provides evidence for at least two decay pathways for the 1-tubulin mRNA. In the deadenylation-dependent constitutive pathway, poly(A) tails must be shortened before the onset of decay, as evidenced by the accumulation of only oligoadenylated intermediates in nondeflagellated cells. In the deadenylation-independent postinduction pathway, poly(A) shortening is not a prerequisite for decay, as evidenced by the accumulation of long poly(A)-tailed intermediates. Both pathways probably involve a 5'-to-3' exonuclease which is impeded by the poly(G) tract. The failure to detect decay intermediates bearing the full range of poly(A) tail lengths in deflagellated cells suggests the occurrence of a second decay pathway that leaves no detectable 3' decay intermediates.