We reported previously the isolation of spontaneous, extragenic suppressors of a temperature-sensitive, lethal poly(A) polymerase mutation (pap1-1
) that restore growth at the nonpermissive temperature 30°C (10
). Characterization of two of these cold-sensitive suppressors, rrp6-1
(previously called pds1-1
]), revealed that the normal, nonessential allele, RRP6
, encodes a putative 3′-5′ riboexonuclease required for efficient 5.8S rRNA 3′-end processing. Since poly(A) tails appear to play a role in enhancing mRNA stability and translation initiation, we compared the ability of a rrp6::URA3
knockout to suppress pap1-1
with knockouts of several genes known to play a role in these processes. The XRN1/SKI1
gene products function in the degradation of mRNAs after deadenylation and during the translation of mRNAs with nonsense codons, respectively (44
). Loss of Xrn1p activity stabilizes mRNAs and leads to the accumulation of uncapped, deadenylated transcripts (21
). Loss of Upf1p leads to the stabilization of mRNAs bearing nonsense codons and suppresses cis
-acting polyadenylation defects in Saccharomyces cerevisiae
and Caenorhabditis elegans
; B. Das, Z. Guo, P. Chartrand, P. Russo, R. Singer, and F. Sherman, submitted for publication). Knockout mutations in either of these genes do not suppress the temperature sensitivity caused by the pap1-1
mutation, while the recessive rrp6-1
mutation, or a knockout of RRP6
, allows pap1-1
cells to grow at 30°C (Fig. ).
FIG. 1 Suppression of pap1-1 temperature sensitivity by mutations in RRP6. Strains BPO2 (PAP1 RRP6), BPO2-12F (PAP1 rrp6::URA3), UR3148-1B (pap1-1), UR3148-1BC-12 (pap1-1 rrp6-1), UR3148-1B-12F (pap1-1 rrp6::URA3), UR3148-1B ΔX (pap1-1 xrn1::URA3), UR3148-1B (more ...)
Deletion of the SPB2(RPL46)
gene encoding the large subunit ribosomal protein L46 causes a decrease in 60S ribosomal subunit levels and bypasses the requirement for the otherwise essential poly(A)-binding protein (Pab1p), presumably by increasing the relative concentration of 40S ribosomes, thereby enhancing the rate of translation initiation of Pab1p-deficient mRNAs at the 40S binding step (16
mutations also cause a decrease in 60S ribosomal subunit levels due to inefficient 5.8S rRNA 3′-end formation (11
). Based on the proposed mechanism for the suppression of pab1
mutations by spb2
, we initially thought that rrp6
mutations might belong to a general class of mutations that bypass the requirement for the poly(A) tail by enhancing the binding of poly(A)−
mRNAs to the 40S subunit. Accordingly, we expected loss of SPB2
to suppress pap1-1
under conditions in which suppression by loss of RRP6
occurs. However, deletion of SPB2
does not suppress the growth defect caused by pap1-1
RRP6 mutations enhance the accumulation of poly(A)+ mRNAs in pap1-1 cells.
Our previous characterization of the effects of polyadenylation shutoff in a pap1-1
strain indicated that the cells die because they fail to accumulate many mRNAs and that inhibition of translation is a secondary consequence of mRNA loss (58
). Thus, suppression of the pap1-1
growth defect under these conditions should result in an increase in steady-state mRNA levels. We quantitated by Northern blot analysis mRNA levels in pap1-1
cells after a shift to 30°C and normalized them to the levels of the stable RNA polymerase III transcript SCR1
. In an effort to avoid secondary effects of Pap1p inactivation, we prepared RNA from cells harvested 2 h prior to growth cessation. Results representative of several experiments show suppression of the mRNA loss phenotype by rrp6-1
, and rrp6::URA3
(Fig. ). Two experiments show that the mRNAs produced in the rrp6
strains carry poly(A) tails. First, visualization of total poly(A) after [5′-32
P]pCp 3′-end labeling and PAGE shows a two- to threefold increase in the amount of poly(A) in an rrp6
strain compared to a pap1-1
strain at 30°C (Fig. ). Second, oligo(dT)-mediated separation of total RNA samples into those with long poly(A) tails (Fig. , lanes 1, 3, 5, and 7) and those with short or no poly(A) tails (Fig. , lanes 2, 4, 6, and 8) reveals that the RRP6
deletion increase two- to threefold the levels of poly(A)+ TCM1
mRNA in pap1-1
cells at 30°C. Both of these experiments detect relatively low levels (ca. 10% of wild type) of poly(A)+
mRNA in pap1-1
cells at 30°C, suggesting that the mutant Pap1p remains partially active at the nonpermissive temperature. This finding contrasts with our previous studies of the effects of the pap1-1
mutation at 37°C, where we detected only unadenylated transcripts (58
FIG. 2 Restoration of mRNA levels in pap1-1 strains caused by mutations in RRP6. (A) Steady-state levels of TCM1 mRNA in rrp6 mutants. Total RNA was isolated from strains with the indicated genotypes before and 6 h after a shift to 30°C. TCM1 mRNA was (more ...)
FIG. 3 RRP6 mutation allows the accumulation of poly(A)+ RNA in pap1-1 strains. Total RNA isolated as in Fig. was 3′-end labeled with 5′-[32P]pCp and T4 RNA ligase. After hydrolysis of non-poly(A) tracts, (more ...)
FIG. 4 RRP6 mutation allows the accumulation of poly(A)+ RNA in pap1-1 strains. Northern blot analysis of total RNA isolated as in Fig. after fractionation on oligo(dT)-cellulose (54). Lanes 1, 3, 5, and 7 contain RNA that was bound to (more ...)
Deletion of UPF1
does not increase the levels of TCM1
transcripts, but it does reproducibly increase the level of ACT1
mRNA (Fig. B). Incomplete resolution of the unspliced and mature forms of the transcript in this experiment leaves open the issue of whether loss of Upf1p preferentially stabilizes unspliced ACT1
mRNA, as it does with other intron-containing mRNAs (30
Deletion of XRN1/SKI1
results in an increase in the levels of all three test mRNAs (Fig. B) but does not increase the levels of poly(A) in a pap1-1
background at 30°C (Fig. , lane 8). Moreover, the XRN1/SKI1
deletion results in a relative increase in TCM1
mRNA that does not bind to oligo(dT)-cellulose, suggesting that this mutation results in the accumulation of unadenylated or deadenylated mRNAs (Fig. , lane 8). Since loss of Xrn1p/Ski1p activity does not suppress the growth defect of pap1-1
cells under these conditions, and based on the accepted role of Xrn1p/Ski1p as the major 5′-3′ riboexonuclease involved in mRNA degradation (37
), we propose that these transcripts accumulate as uncapped, deadenylated intermediates of the normal mRNA decay pathway and that the lack of a cap or poly(A) tail on these transcripts likely results in their inefficient translation.
Rrp6p does not play a role in the rate-limiting step of mRNA decay.
The increase in poly(A)+ mRNA levels in the absence of Rrp6p and the homology of the enzyme to E. coli 3′-5′ RNase D (see below) suggest that loss of Rrp6p activity could suppress the pap1-1 defect if the enzyme played a role in the rate-limiting step of poly(A)+ mRNA decay. We tested this hypothesis by measuring the decay rates of several mRNAs after inhibition of RNA polymerase with the drug thiolutin. Northern blot and graphical analyses of TCM1 mRNA decay rates show that the loss of Rrp6p function does not decrease mRNA decay rates (Fig. ; Table ). These experiments were carried out with several mRNAs in a PAP1 background and in a pap1-1 background after a shift to 30°C (Table ); in neither case did the rrp6::URA3 knockout slow the mRNA decay rates. Differential sensitivity of distinct strains to thiolutin could conceivably result in incomplete inhibition of transcription in some strains, resulting in incorrect decay rate values. We do not believe that this problem affects our measurements of the effects of deletion of RRP6 on decay rates since continued transcription would cause an apparent decrease in the measured rate of decay, which we did not observe. We conclude that Rrp6p does not play a role in the rate-limiting step of mRNA degradation and that suppression of the pap1-1 mRNA accumulation defect by rrp6 mutations may occur at a step prior to the major mRNA decay pathway.
FIG. 5 TCM1 mRNA decay rates in RRP6 and Δrrp6 cells. Shown are the results of a Northern blot analysis of TCM1 mRNA levels in total RNA samples from cells as a function of time after treatment with the transcriptional inhibitor thiolutin. The graph (more ...)
TABLE 3 Effect of RRP6 mutation on mRNAhalf-lives Nuclear localization of Rrp6p.
Since Rrp6p defects increase poly(A)+ mRNA levels without slowing mRNA decay, we surmised that the enzyme may play a role in limiting the concentration of unadenylated mRNAs at an early step in mRNA biogenesis. Since mRNA maturation, including polyadenylation, occurs in the nucleus, we determined the subcellular localization of Rrp6p. We fused GFP to the amino terminus of Rrp6p and expressed this fusion from a low-copy plasmid in a strain carrying an rrp6::KAN knockout. Expression of the fusion protein in these cells suppresses both the temperature sensitivity and the 5.8S rRNA processing defect caused by the rrp6::KAN mutation, indicating that the fusion protein functions as Rrp6p (data not shown). Fluorescence microscopy of logarithmically growing cells expressing GFP-Rrp6p and comparison with nuclear DNA staining of the same cells shows that the majority of the protein resides in the nucleus (compare Fig. A and B). GFP alone distributes itself evenly between the nucleus and the cytoplasm, but it is excluded from the vacuoles, as expected (Fig. C). Although this experiment does not exclude the possibility that some small fraction of Rrp6p resides in the cytoplasm, it does suggest, along with the protein's role in rRNA processing, that its major activity likely takes place within the nucleus.
FIG. 6 Subcellular localization of GFP-Rrp6p in logarithmically growing yeast cells. Strain BPKAN carrying plasmid pGFP-RRP6-FOR11 (A and B) or pGFP-RRP6-REV2 (C) were grown in synthetic complete medium lacking uracil at 30°C to a density of approximately (more ...) RRP6 mutation causes a decrease in LA RNA levels.
Many strains of S. cerevisiae
harbor a dsRNA virus (LA virus) whose life cycle takes place in the cytoplasm (68
). LA virus produces uncapped, unadenylated mRNAs that compete with cellular mRNAs during translation initiation. Like cellular mRNAs, the concentration of LA mRNAs is regulated by the cytoplasmic 5′-3′ riboexonuclease encoded by XRN1/SKI1
). Mutation of host SKI
genes such as XRN1/SKI1
, as well as SKI6/RRP41
encoding the exosomal 3′-5′ riboexonuclease Rrp41p, causes an increase in the expression and the amount of LA RNA (8
). In contrast, mutations in host MAK
genes, many of which affect ribosomal subunit biogenesis, cause a decrease in LA RNA levels (24
). We reasoned that if Rrp6p plays a role in degrading unadenylated mRNAs in the cytoplasm, then rrp6
mutants should display an SKI
phenotype. We tested this by Northern blot analysis of the levels of LA RNA in rrp6-1
strains and in the same strains complemented by a plasmid-borne copy of RRP6
. Instead of an SKI
phenotype, loss of Rrp6p activity causes a decrease in LA RNA levels reminiscent of an MAK
phenotype (Fig. ). We suggest that the decrease in LA RNA levels, like that associated with many MAK
mutations, results from the decrease in the 60S/40S subunit ratio caused by rrp6-1
). These findings, along with the nuclear localization of Rrp6p demonstrated above, support a nuclear role for Rrp6p in RNA processing.
RRP6 mutations cause a decrease in LA RNA levels. Northern blot analysis of LA RNA levels from two different rrp6-1 strains carrying the indicated plasmids is shown. LA RNA levels were normalized to SCR1 RNA levels as indicated below.
Rrp6p is a 3′-5′ riboexonuclease.
The homology of Rrp6p to the E. coli
3′-5′ riboexonuclease RNase D and the fact that rrp6
mutations result in the accumulation of a 3′-extended form of 5.8S rRNA led us to suggest a 3′-5′ riboexonuclease activity for Rrp6p (10
). Figure shows the homology of the core region of Rrp6p with that of a representative group of related proteins, including E. coli
RNase D and the 3′-5′ deoxyriboexonuclease domain of E. coli
DNA polymerase I (7
). Mian and colleagues have pointed out that enzymes of the RNase D class have domains whose sequence and spatial conservation resemble the catalytic domain required for the 3′-5′ deoxyriboexonuclease activity of DNA polymerase I (46
). Based on enzymatic analysis of specific amino acid changes in these domains and comparison with the enzyme's crystal structure, Steitz and colleagues proposed a two-metal ligand mechanism for phosphodiester bond cleavage that features the coordination of nucleophilic metal ions by specific amino acid side chains in these domains (7
). Nucleotide sequence analysis of rrp6-1
shows that it contains an aspartate-to-asparagine mutation at position 238 which, based on the two-metal ligand mechanism, would inactivate the exonuclease activity of the enzyme. Indeed, the rrp6-1
mutation leads to the accumulation of 3′-extended 5.8S rRNA molecules, a finding consistent with loss of the enzyme's 3′-5′ riboexonuclease activity (10
FIG. 8 Comparison of the predicted catalytic core of Rrp6p with homologues from Homo sapiens (PM-Scl 100 kDa; Q01780), Schizosaccharomyces pombe (Q10146), C. elegans (P34607), and E. coli (RNase D, P09155; POL, P00582). Amino acid identities occurring in five (more ...)
We tested Rrp6p for exonuclease activity by purifying a GST-Rrp6p fusion after expression in E. coli and by incubating it with different 5′-32P-labeled RNAs (Fig. ). Expression of this GST-Rrp6p fusion in yeast complements the growth defects of an rrp6::KAN mutant, indicating normal function of the fusion protein in vivo (data not shown). Incubation of GST-Rrp6p with a 5′-32P-labeled CYC1 pre-mRNA substrate results in the production of specific degradation products, while GST alone shows little degradation of the substrate (Fig. B). The production of increasingly shorter 5′-32P-labeled CYC1 pre-mRNA products as a function of time and the fact that we observe this same pattern of intermediates when the substrate carries 5′-terminal cap (data not shown) imply a 3′-5′ directionality for the enzyme. A different 5′-32P-labeled RNA substrate yields a similar pattern of hydrolysis (Fig. C; lanes 1 to 4). However, placement of a 3′ PO4 at the RNA's 3′ end by ligation of 5′-[α-32P]pCp inhibits hydrolysis by GST-Rrp6p (Fig. C, lanes 5 to 8). Finally, we incubated GST or GST-Rrp6p with an internally labeled RNA substrate synthesized with SP6 RNA polymerase, unlabeled nucleotide triphosphates, and 5′-[α-32P]GTP (Fig. D). Analysis of the reaction products by thin-layer chromatography and comparison to nucleotide monophosphate standards show that Gst-Rrp6p produces a single radiolabeled product that comigrates with 5′pG, the product expected of a hydrolytic exonuclease (Fig. D, lanes 5 and 6). Taken together, these findings indicate that Rrp6p hydrolyzes RNA substrates by a 3′-5′ exonucleolytic mechanism. Unfortunately, we were unable to test the activity of the rrp6-1 mutant protein since it is unstable when expressed in E. coli.
FIG. 9 Exonuclease activity of recombinant Rrp6p. (A) SDS-PAGE analysis of GST-Rrp6p and GST. After gel electrophoretic separation, the gel was stained with SYBRO RED (Molecular Probes) and analyzed by fluorimager analysis. (B) Storage phosphorimager analysis (more ...) Rrp6p interacts with poly(A) polymerase and the hnRNA protein Npl3p.
In an effort to extend our understanding of the role of Rrp6p in mRNA processing, we searched for proteins that interact with Rrp6p by using the two-hybrid screen (4
). We fused Rrp6p to the DNA binding domain of Gal4p (GDB-RRP6) and screened a library of Gal4p-activation domain fusions (GAD) for their ability to activate transcription of GAL1-lacZ
reporters. Of 58,000 transformants screened, 20 grew in the presence of 3-AT and produced blue colonies in the presence of X-Gal, indicating the expression of GAL1-HIS3
, respectively. DNA sequence analysis of the inserts in the candidate plasmids revealed 14 different genes, including one example of NPL3
. We chose NPL3
for further study because it is thought to function as an hnRNA protein involved in mRNA processing and transport of mRNA out of the nucleus (43
). Moreover, NPL3
interacts directly with mRNA and genetically with RNA15
, which encode proteins directly involved in mRNA 3′-end processing (31
). Neither GDB-RRP6 nor GAD-NPL3 activates transcription of GAL1-HIS3
on its own, as evidenced by the inability of reporter strains carrying either of these plasmids to grow in the presence of 100 mM 3-AT (Fig. A and data not shown). Each of these fusions also fails to promote growth under these conditions in the presence of other fusions, including, in the case of GDB-RRP6, the exosomal riboexonuclease GAD-RRP43 (Fig. A) (48
). These findings suggest a specific interaction between Rrp6p and Npl3p in vivo (Fig. A).
FIG. 10 Interaction of Rrp6p with Npl3p and Pap1p. (A) Growth at 30°C of strains carrying various GAL4 DNA binding domain (GBD) fusions and GAL4 activation domain (GAD) fusions on plates with or without the addition of 100 mM 3-AT. The diagram in the (more ...)
We sought further evidence for an interaction between Rrp6p and Npl3p by assaying for copurification of the two proteins. We constructed a plasmid capable of expressing in yeast cells the same GST-Rrp6p fusion protein used in the experiments illustrated in Fig. , and we showed that this plasmid complements the growth defect caused by an RRP6 knockout (data not shown). Affinity purification of GST-Rrp6p from yeast cells resulted in copurification of Npl3p, as judged by Western blot analysis (Fig. B, lane 4). GST was efficiently purified by this procedure, but Npl3p did not copurify with it (Fig. B, lane 2). Moreover, a negative control large subunit ribosomal protein, Tcm1p, does not copurify with GST or GST-Rrp6p (Fig. B, lane 2). These results confirm that Rrp6p and Npl3p interact specifically in vivo.
Next, we investigated the functional consequences of disrupting the interaction between Rrp6p and Npl3p. We knocked out the chromosomal allele of RRP6 in a strain carrying a chromosomal npl3-1 mutation and a plasmid-borne RRP6 allele. Plating such cells on 5-fluoroorotic acid (5 FOA) selects for those that have lost the plasmid and reveals that the combination of the npl3-1 mutation and the RRP6 deletion is lethal (Fig. C). Since cells carrying either of these mutations alone survive this test, we conclude that the mutations are synthetically lethal and that Rrp6p and Npl3p interact functionally in vivo.
Because our findings indicated that Rrp6p may function to degrade unadenylated mRNAs and that it exists as part of a complex containing Npl3p, we asked whether Rrp6p also interacts with Pap1p. The affinity purification of Rrp6p illustrated in Fig. B shows that Pap1p also copurifies with Rrp6p. We did not identify PAP1
as a result of our two-hybrid screen with GDB-Rrp6p, and others have also failed to identify interaction of Rrp6p with Pap1p by similar methods (22
). Thus, Pap1p and Rrp6p may interact together indirectly. Nevertheless, copurification of Rrp6p and Pap1p supports our model, based on the findings presented above, that Rrp6p may function as part of a complex of proteins that monitors the polyadenylation step of mRNA 3′-end processing such that it degrades mRNAs that fail to be polyadenylated.