To identify additional functions for the exosome complex, we created a collection of eight strains carrying mutations in different components of the exosome, or in the proteins related to exosome function (Table ), and then examined a diversity of 3′-to-5′ processing reactions in that collection of strains. Three strains that contained conditional temperature-sensitive alleles in core components of the exosome (i.e., rrp4-1
, and mtr3-1
) were used. These core components are thought to be subunits of both the nuclear and the cytoplasmic exosome (1
) and are all essential. In addition, a strain carrying a deletion of RRP6
was examined. Rrp6p is an exonuclease associated with the exosome in nuclear fractions (1
), and rrp6
Δ strains have a defect in 5.8S rRNA maturation (2
). A temperature-sensitive mtr4-1
strain was also included. Mtr4p is an essential protein required for 5.8S rRNA processing by the exosome (9
). Last, strains with deletions of the nonessential SKI2
, and SKI8
genes were examined. These three genes are known to be required for exosome-mediated 3′-to-5′ degradation of mRNA (13
Yeast strains containing a deletion of a nonessential gene (SKI2, SKI3, SKI8, or RRP6) were grown at 30°C. Therefore, the pool of stable RNA in these strains represent a steady-state condition. In contrast, yeast strains containing conditional mutations in essential genes (RRP4, SKI6, MTR3, and MTR4) were grown at 24°C (a temperature permissive for growth) and shifted to 37°C (a temperature restrictive for growth) for 1 h. A relatively short shift to the restrictive temperature was used to minimize the occurrence of secondary effects. As a consequence, the pool of stable RNA present is a mixture of RNA synthesized during growth at the permissive temperature and RNA synthesized at the restrictive temperature. A wild-type strain was grown under both conditions. Total RNA from wild-type and mutant strains was analyzed by Northern blotting using oligonucleotide probes for a wide variety of RNA species. These included rRNA, tRNA, mRNA, snRNA, snoRNA, and the RNA subunits of SRP and RNases P and MRP. This collection of RNAs includes species transcribed by all three RNA polymerases and includes transcripts previously suggested to be processed from 3′-extended precursors, transcripts whose mature 3′ end appears to match the polymerase termination signal, and species for which little, if anything, is known about 3′ end formation.
Importantly, as detailed below, examination of U4 snRNA and snoRNAs revealed several alterations in these transcripts in the ski6-100, rrp4-1, rrp6Δ, mtr3-1, and mtr4-1 mutant strains but not in the ski2Δ, ski3Δ, or ski8Δ strains. In contrast, to date we have not found obvious defects in the processing of many other RNAs, including 5S rRNA, several tRNAs, and the RNA subunits of SRP and RNases MRP and P in any of the mutant strains examined (data not shown). This observation is consistent with these particular RNA species having mature 3′ ends that are formed by transcriptional termination, by processing by other 3′-to-5′ exonucleases (A. van Hoof, P. Lennertz, and R. Parker, unpublished data), or by redundancy of the exosome with other exonucleases for these functions. In either case, these results serve as negative controls that indicate that the defects described below are specific to U4 snRNA and snoRNAs.
Exosome mutants accumulate 3′-extended forms of snoRNAs and U4 snRNA. (i) The exosome is involved in the processing of independently transcribed snoRNAs and U4 snRNA.
snoRNAs are produced from independent transcripts, excised introns, or polycistronic transcripts (reviewed in references 20
). To examine the role of the exosome in the processing of independently transcribed snoRNAs, we examined a member of each of the two major classes of snoRNAs (Table ). snoRNAs can structurally and functionally be divided into C/D box-containing snoRNAs, required for methylation of the 2′ hydroxyl of RNA, and H/ACA box-containing snoRNAs, required for pseudouridyl formation in rRNA. For each class, we examined one snoRNA transcribed from an independent transcription unit. For the C/D box-containing snoRNAs, we examined snR40; for the H/ACA box-containing snoRNAs, we examined snR33.
Two sets of observations suggest that the exosome is involved in the processing of 3′-extended forms of snR33 and snR40. First, in rrp6Δ strains, the majority of snR33 and snR40 RNA species were longer by a few nucleotides (Fig. A and C, lower panels). This observation suggested that the exosome, and perhaps Rrp6p specifically, is involved in the removal of the last few 3′ nucleotides, to give rise to the mature 3′ end of these RNAs. The second alteration was that rrp6Δ, mtr4-1, ski6-100, mtr3-1, and rrp4-1 mutant strains all accumulated longer heterogeneous forms of the snR33 and snR40 transcripts (Fig. A and C, top panels). Hybridization of a Northern blot containing RNA from wild-type and rrp6Δ strains with a probe designed to hybridize 3′ of the mature snR33 showed that the heterogeneous population seen in Fig. A represents 3′-extended forms of snR33 (Fig. B). These observations suggest that snR33 and snR40 are made as 3′-extended forms that are then processed in a manner requiring the exosome (see below).
FIG. 1 Exosome mutants have defects in processing of independently transcribed snoRNAs and U4 snRNA. Shown are two different exposures of the same Northern blots containing RNA from the indicated strains. All strains were grown in YEP–2% galactose (more ...)
Mutations affecting exosome function, especially mtr4-1
, and rrp4-1
, led to only modest accumulation of the heterogeneous 3′-extended forms of snoRNAs. However, these defects are likely to be biologically significant for four reasons. First, snoRNAs are thought to be stable, and thus the pool of snoRNAs in a temperature-sensitive mutant after 1 h at the restrictive temperature reflects mostly RNA produced before the shift to the restrictive conditions. Second, the defects seen here are similar in strength to those seen in rRNA processing and mRNA degradation in the same mutants. For example, mutations in RRP4
led to only modest increases in 3′-extended 5.8S rRNA, which were detected only by long exposures or with use of probes designed not to hybridize to the mature RNA (9
). Third, the defects seen here are similar in strength to defects in snoRNA 5′ processing in a rat1-1 xrn1
Δ double mutant. This mutant accumulates low levels of 5′-extended forms of snR190, U14, U24, and U18 snoRNAs after a 2-h incubation at the restrictive temperature (27
), some of which were shown to exist only by using probes specific for 5′-extended forms. Fourth, reproducible accumulation of heterogeneous 3′-extended species were seen for six different snoRNAs from different classes (see below).
Analysis of the independently transcribed U4 snRNA revealed results similar to but distinct from those for snR33 and snR40. Wild-type strains accumulated a mature U4 species of 160 nucleotides (nt) and 3′-extended forms of between 270 and 300 nt. In addition, rrp6Δ, mtr3-1, mtr4-1, ski6-100, and rrp4-1 strains accumulated RNA species intermediate in size between the mature species and the 270- to 300-nt species. A heterogeneous population of transcripts was also seen in the rrp6Δ strain extending above the 270- to 300-nt precursor (Fig. D). The use of a specific oligonucleotide probe 3′ of the mature 3′ end of the U4 RNA showed that these species were extended on the 3′ side of the RNA (Fig. E). These data suggest that the exosome is involved in the processing of the 3′-extended forms of U4 snRNA.
(ii) The exosome is involved in the processing of intron-derived snoRNAs.
To determine if the exosome functions in the processing of intron-derived snoRNAs, we examined a member of each of the two major classes of snoRNAs. For the C/D box-containing snoRNAs, we examined U24; for the H/ACA box-containing snoRNAs, we examined snR44.
Intron-derived snoRNAs also showed altered patterns of accumulation in the various strains examined. First, for U24 in the rrp6Δ strain, the predominant RNA species was slightly larger than the normal RNA (Fig. A). This observation is similar to that noted above for the independently transcribed snoRNAs snR33 and snR40, and it suggests that the exosome, and perhaps Rrp6p specifically, is involved in the removal of the last few nucleotides to give rise to the mature 3′ end of these transcripts. Second, we observed a slight but reproducible reduction in the level of snR44 in the various mutants. The residual snR44 levels varied from 10% of the wild-type level in the rrp6Δ strain to 75% for the mtr4-1 strain (Fig. B). Less drastic reductions were seen for the level of U24 in various exosome mutants (Fig. A). One possible interpretation of these observations is that in the absence of proper 3′-to-5′ trimming, snR44 is degraded.
FIG. 2 Exosome mutants have defects in processing of intron-derived snoRNAs. Shown are Northern blots containing RNA from the indicated strains. All strains were grown in YEP–2% galactose to early to mid-log phase. The ski2Δ, ski3Δ, (more ...)
We also examined the effects of exosome mutations on the processing of the U18 snoRNA. This snoRNA is embedded within the intron of the EFB1
gene, but it has been shown to be processed by two different pathways (36
). In one pathway, it is processed from an intron released from the EFB1
pre-mRNA; in the other pathway, it is processed from the primary transcript in a manner that does not require splicing. Thus, this snoRNA does not fit neatly in either the intron-derived or the independently transcribed snoRNA class.
The defects seen for U18 snoRNA were similar to those seen for other snoRNAs. First, the rrp6Δ strain primarily accumulated a U18 RNA that was slightly larger than normal, as well as a heterogeneous population of larger species. A strain containing the mtr3-1 mutation (and possibly mtr4-1, ski6-100, and rrp4-1 strains) accumulated low levels of heterogeneous species of 200 to 300 nt (Fig. C). Probing of a Northern blot with RNA from wild-type and rrp6Δ strains with an oligonucleotide probe 3′ of the mature 3′ end of the U18 snoRNA showed that the heterogeneous species in rrp6Δ were extended on the 3′ side of the RNA (Fig. D). The observation that essentially no mature U18 accumulates in an rrp6Δ strain suggests that both pathways of U18 maturation are affected. Based on the defects seen in rrp6Δ, combined with the slight defects seen in other exosome mutants, we conclude that Rrp6p, and presumably also the core exosome, is involved in both pathways of U18 processing.
The exosome is involved in the processing of polycistronic snoRNAs.
To examine the role of the exosome in the processing of polycistronic snoRNAs, we examined two polycistronic precursors. One transcript examined contains snR190 and U14 C/D box-containing snoRNAs, with U14 being the 3′ snoRNA (6
). The second polycistronic transcript examined contains seven C/D box-containing snoRNAs, in the 5′-to-3′ order of snR78, snR77, snR76, snR75, snR74, snR73, and snR72 (28
). No H/ACA box-containing snoRNAs derived from polycistronic transcripts have been described to date.
Exosome mutants showed two distinct defects in the processing of snoRNAs from polycistronic precursors. Similar to what was seen with other snoRNAs, the rrp6Δ strain accumulated slightly larger than normal transcripts for U14, snR73, and snR72 (Fig. B to D, lower panels). No difference was seen for snR190 (Fig. A), but a difference of one or a few nucleotides might not be resolved, due to the relatively large size of this snoRNA. While snR33 is similar in size to snR190, we were able to detect an effect of rrp6Δ on snR33; thus, if rrp6Δ results in a larger snR190, this size difference must be smaller than the corresponding size difference for snR33. In addition to this effect, rrp6Δ, mtr3, mtr4, ski6, and rrp4 mutant strains accumulated heterogeneous populations of RNAs that were 3′-extended forms of U14, snR73, and possibly snR72 but not of snR190 (Fig. ). These data, combined with the defects seen for independently transcribed and intron-derived snoRNAs, suggest that the exosome is involved in the processing of 3′-extended snoRNAs, irrespective of whether they are derived from independent transcripts, introns, or polycistronic transcripts.
FIG. 3 Exosome mutants have defects in processing of polycistronic snoRNAs. Shown are two different exposures of the same Northern blots containing RNA from the indicated strains. All strains were grown in YEP–2% galactose to early to mid-log (more ...) Exosome mutants accumulate polyadenylated forms of U4 snRNA and some snoRNAs.
In the rrp6
Δ mutant and in several of the other exosome mutant strains, we observed a larger heterogeneous population of transcripts derived from U4 snRNA and snR33, snR40, U18, U14, snR73, and snR72 snoRNAs. The simplest explanation is that this population represents a pool of polyadenylated RNAs with poly(A) tails varying in length. To examine this possibility, two experiments were performed. First, RNAs from different mutant strains and a wild-type strain were treated with RNase H in the presence of oligo(dT). RNase H cleaves RNA in a RNA-DNA duplex and is commonly used in combination with oligo(dT) to remove poly(A) tails in vitro. The products of these RNase H reactions were analyzed by probing a Northern blot for snR33. An important observation was that following treatment with RNase H and oligo(dT), the heterogeneous population migrated faster in the gel. Since the largest stretch of A's encoded in the corresponding region of the genome (three A's) is not long enough to efficiently hybridize to oligo(dT), this RNase H induced shift is not caused by a stretch of encoded A's and thus must be the result of a poly(A) tail. Removal of the poly(A) tail by RNase H treatment did not result in one discrete band, presumably because the poly(A) tail in individual molecules was added at different sites. This is similar to what occurs with mRNAs. For example, Graber et al. (11
) recently analyzed 1,352 unique mRNA 3′ ends, which they found to be derived from 861 genes. Thus, even in this small sample, the average number of 3′ ends per gene was 1.6, indicating that many genes produce mRNAs with alternative 3′ ends. Our results indicate that the heterogeneous population seen in untreated samples corresponded to polyadenylated snoRNAs. These polyadenylated snR33 species are present at low levels in the wild type (Fig. A), and these levels are elevated in at least four strains carrying exosome mutations (Fig. B).
FIG. 4 Wild-type and exosome mutant strains accumulate polyadenylated snR33. Shown is a Northern blot containing RNA from the indicated strains. All strains were grown in YEP–2% galactose to early to mid-log phase. The rrp6Δ strain was (more ...)
Similar experiments were also done to test whether the heterogeneous populations seen for snR40, U4, U18, U14, snR73, and snR72 in the rrp6Δ strain correspond to polyadenylated RNAs. Upon RNase H and oligo(dT) treatment, the heterogeneous population of 3′-extended species for U18, U14, snR73, and snR72 collapsed into a faster-migrating species (Fig. C to F, compare rrp6 lanes to rrp6 +dT lanes). While snR40 and U4 snRNA did not collapse into a single band upon RNase H and oligo(dT) treatment, the heterogeneous species for these two RNA migrate faster after this treatment, indicating that these species were also cleaved. Because the genomic regions just 3′ of these RNAs also do not contain long stretches of A's, this RNase H-induced shift must also be the result of a poly(A) tail. Thus, the heterogeneous species of all six RNAs are polyadenylated. As a negative control, the same analyses were done with snR190 and U24, which did not accumulate as heterogeneous populations. RNase H and oligo(dT) treatment did not result in a change in the pattern seen for either snR190 or U24 (Fig. G and H), suggesting that these two snoRNAs are not polyadenylated.
FIG. 5 The rrp6Δ mutant accumulates polyadenylated forms of snR40, U4, U18, U14, snR73, and snR72 but not U24 or snR190. Shown are Northern blots containing RNA from the indicated strains. All strains were grown in YEP–2% galactose to (more ...)
The second experiment to address whether the longer species were polyadenylated was to examine their presence in an rrp6Δ pap1-1
double-mutant strain. The PAP1
gene encodes the yeast poly(A) polymerase. The pap1-1
allele confers a temperature-sensitive defect in the addition of poly(A) tails to mRNA (25
). Northern blot analysis of RNA from an rrp6Δ pap1-1
strain grown at 24°C showed that this strain accumulated the heterogeneous 3′-extended transcripts, but they were shorter than those in the rrp6
Δ control strain (Fig. A to F, compare rrp6 lanes to rrp6 pap1-1 24C lanes). This indicates a partial defect in polyadenylation, even at the permissive temperature. More important, the heterogeneous population disappeared after a 1-h shift to 37°C (the restrictive temperature for pap1-1
; Fig. A to F, compare rrp6 and rrp6 pap1-1 24C lanes to rrp6 pap1-1 37C lanes), indicating that Pap1p is required for the accumulation of this heterogeneous population.
We interpret the above data to suggest that at least a portion of the primary transcripts of snoRNAs are produced as polyadenylated species utilizing Pap1p and that the exosome functions to deadenylate these RNAs (see Discussion). Interestingly, the rrp6Δ pap1-1 double mutant did not accumulate abundant 3′-extended nonpolyadenylated snoRNAs that would comigrate with the species seen in the rrp6Δ +dT lanes. This finding indicates that while rrp6 mutants deadenylate these species slowly, further 3′ trimming is not as strongly affected. Finally, the polyadenylated snoRNA species that accumulated in the rrp6Δ pap1-1 mutant at 24°C disappeared within 1 h after inactivation of Pap1p. This observation suggests that this strain still contains an activity that can process, or degrade, the polyadenylated snoRNA species (see below for further discussion).
Rnt1p and the exosome act in the same pathway of U4 snRNA processing.
Recently it has been shown that Rnt1p, a double-strand-specific endoribonuclease, processes the U4 snRNA at two sites located 135 and 169 nt 3′ of the mature 3′ end, generating precursors of approximately 300 nt (1a
). This is the same size as the 3′-extended U4 RNA that we detect in our Northern blots and that accumulates as a polyadenylated species in the rrp6
Δ strain. These observations suggest two mechanisms for the formation of the polyadenylated U4 RNAs. In one model, the 3′-extended forms of the U4 snRNA would be made by the assembly of the normal mRNA polyadenylation machinery onto the nascent transcript, which would then lead to cleavage and polyadenylation. This would generate polyadenylated transcripts similar in size to the Rnt1p cleavage products but independently of Rnt1p. Alternatively, following Rnt1p cleavage, the 3′-extended U4 snRNA could be a substrate for polyadenylation by Pap1p. This would be striking since it would imply that the poly(A) polymerase can function on a second class of substrates cleaved by a distinct endonuclease. This latter hypothesis predicts that the formation of the polyadenylated U4 RNAs should be dependent on the Rnt1p.
To test this prediction, we examined the processing of the U4 snRNA in rnt1Δ and rnt1Δ rrp6Δ double-mutant strains. As expected, the rnt1Δ strain lacked the approximately 300-nt-long 3′-extended forms of U4 snRNA that are normally seen in wild-type cells. More important, in the rnt1Δ rrp6Δ double mutant strain the 3′-extended U4 snRNA species normally seen in rrp6 strains disappeared. Instead, a new heterogeneous species of >500 nt that may itself be polyadenylated appeared in the double mutant (Fig. A). These data strongly suggest that the polyadenylated U4 RNAs are produced by the addition of poly(A) tails to precursors that were cleaved by Rnt1p (see Discussion). For comparison, the processing of U18 snoRNA was analyzed in the rnt1Δ rrp6Δ strain. Rnt1p has no known role in U18 snoRNA processing, and the rnt1Δ mutation did not affect U18 processing (Fig. B) or prevent the accumulation of polyadenylated U18 species in the rrp6Δ strain, although the abundance of the U18 polyadenylated species was slightly reduced.
FIG. 6 Rnt1p and Rrp6p act in the same pathway of U4 snRNA processing. Shown are Northern blots containing RNA from the indicated strains. All strains were grown in YEP–2% glucose to early to mid-log phase at 24°C. Blots were probed for (more ...) Effects of mutations in exosome components on mRNA degradation.
Since the exosome is involved in deadenylation of snoRNAs, as well as in 3′-to-5′ degradation of mRNA, the exosome might also function in the cytoplasmic deadenylation of mRNA. To examine this possibility, we measured the deadenylation rate of the MFA2pG and PGK1pG mRNAs (11a
) by transcriptional pulse-chase analyses (8
). In this analysis, a short pulse of transcription is used to produce mRNA with long poly(A) tails. The rate of deadenylation can then be determined in a chase period following transcription shutoff. This analysis indicated that ski2
, and mtr3-1
mutants shortened the poly(A) tail on both PGK1 and MFA2 at rates indistinguishable from those for the wild type (data not shown). This finding is consistent with prior observations that overall mRNA decay rates are not substantially altered in exosome mutants (data not shown and reference 13
) and argues that the exosome is not required for cytoplasmic deadenylation, although it might be functionally redundant with other 3′-to-5′ exonucleases in the cytoplasm.
We also determined the possible role of different exosome subunits in 3′-to-5′ mRNA degradation of the body of the transcript following deadenylation. A simple assay for 3′-to-5′ degradation of mRNA is to assess the levels and integrity of a poly(G)-to-3′-end fragment from the MFA2pG transcript (13
). This fragment is produced by the 5′-to-3′ mRNA decay pathway. Because it is normally degraded by the exosome, it accumulates in mutants defective in 3′-to-5′ decay of mRNA (13
). As described by Jacobs Anderson and Parker (13
Δ, or ski6-100
resulted in the appearance of a ladder of partially degraded mRNA fragment (Fig. ). In contrast, the rrp6
Δ and mtr4-1
mutants accumulated MFA2pG mRNA degradation intermediates at levels similar to those for the wild type, indicating that Rrp6p and Mtr4p are not required for 3′-to-5′ degradation of mRNA.
FIG. 7 mRNA degradation phenotypes of exosome mutants. Shown is a Northern blot containing RNA from the indicated strains. All strains were grown in YEP–2% galactose to early to mid-log phase. The ski2Δ, ski3Δ, ski8Δ, (more ...)
We also genetically tested the involvement of Rrp6p in 3′-to-5′ decay of mRNA. This test is based on the observation that mutations that block 5′-to-3′ decay of mRNA (such as deletion of the gene encoding the decapping enzyme, DCP1) are synthetically lethal with mutations that block the alternative 3′-to-5′ pathway (13
). A dcp1
Δ strain was crossed with an rrp6
Δ strain, and tetrads from the resulting diploid were dissected. Although both single-mutant strains grow slowly, the double mutant was recovered at the expected frequency and did not show an additional growth defect (data not shown). Similarly, an mtr4-1
mutant was crossed to a dcp1
Δ strain. The double mutants were recovered at the expected frequency from this cross, and these double mutants did not show a more severe growth defect at any temperature tested (23 to 36°C). Thus, both genetic and molecular data indicate that the exosome and the Ski2p, Ski3p, and Ski8p are required for 3′-to-5′ degradation of mRNA but Rrp6p and Mtr4p are not (see Discussion).