It was previously determined that GCR3
, which encodes yCBP80, and MUD13
, which encodes yCBP20, are not essential genes in S. cerevisiae
), strongly suggesting that yCBC is not necessary for yeast vegetative growth. To further investigate this, the growth of yeast strains lacking GCR3
-Δ) or both genes (cbp20/80
-Δ) was analyzed. As shown in Fig. , strains that lack either CBP80 or CBP20 individually grow slowly at 30°C on rich medium either on plates or in liquid culture. The growth defects are similar at higher (37°C) or lower (23°C) temperatures (data not shown). Analysis of extracts made from the strains by Western blotting showed that while the cbp20
-Δ strain accumulated amounts of yCBP80 similar to those accumulated by the wild-type strain, the cbp80
-Δ strain accumulated fourfold less yCBP20 than the wild type, suggesting that yCBP20 is unstable in the absence of yCBP80 (data not shown). Since CBP80 and CBP20 need to heterodimerize to bind to capped RNA (30
) it was not surprising that the two single-deletion strains showed similar growth defects. It was unexpected, however, that a strain lacking both yCBP80 and yCBP20 (cbp20/80-
Δ) grew better than strains with either single deletion (Fig. ). This suggested that the production of either CBP80 or CBP20 alone had a dominant negative effect on growth.
Genetic interactions between yCBC and splicing factors. MUD13
, which encodes yCBP20, was characterized on the basis of a mutant allele that caused synthetic lethality when present in combination with an otherwise viable mutant form of U1 snRNA (10
). This finding, together with biochemical data (10
), showed that yCBC functioned in the commitment complex assembly step of yeast pre-mRNA splicing. Commitment complexes form on intron-containing pre-mRNAs in the absence of ATP hydrolysis. There are two commitment complexes, CC1 and CC2, both of which depend on U1 snRNP-5′ splice site interaction (64
). In addition, CC2 requires interaction between Mud2p and branch point binding protein (BBP), which bind at and near the branchpoint region of the intron, and U1 snRNP bound at the 5′ splice site (1
). The identities of genes complementing LUC1 to LUC6 are consistent with the function of yCBC in commitment complex assembly.
LUC1, LUC2, and LUC6 were initially assigned to this category. LUC1/MUD1
encodes the yeast U1A homologue and also causes synthetic lethality with the truncated U1 snRNA used to identify MUD13
). U1A is a conserved component of U1 snRNP. LUC2/MUD2
was also found in the truncated U1 snRNA SL screen and encodes the yeast homologue of U2AF65 (1
). Both U2AF65 and Mud2p are involved in very early steps of intron recognition (1
). LUC6 is complemented by SMD3
, which encodes one of the core components of the spliceosomal snRNPs (60
; see also reference 47
). Although not specific for U1 snRNP, in fact an SMD3
allele that causes synthetic lethality together with a mutant U2 snRNA has previously been isolated (81
); mutation of SmD3p could be expected to affect U1 snRNP function at early stages of splicing.
The product of NAM8
, which complements LUC3, was originally proposed to have a role in mitochondrial RNA splicing (13
) and later implicated in meiosis-specific nuclear pre-mRNA splicing events (54
). Recently, however, it was identified, along with the products of SNU56/MUD10
, which complements LUC4, and SNU71
, which complements LUC5, as a novel component of the yeast U1 snRNP (21
). These three proteins are all stably associated with yeast U1 snRNA but are not present in vertebrate U1 snRNP (14
). Since yCBC and U1 snRNP are both commitment complex components, these findings provide a reasonable explanation for the synthetic lethality that results when these three genes are mutated on a yCBC null background.
, which complements LUC7, is not functionally characterized. We have found putative vertebrate homologues by examination of the DNA databases. These homologues have SR domains, characteristic of a large family of metazoan splicing factors (59
), consistent with the possibility that the LUC7 SL phenotype is also due to mutation of a protein involved in pre-mRNA splicing. Further characterization of this complementation group is in progress.
yCBC deletion strains exhibit defects in rRNA processing.
LUC8 (two strains) and LUC9 (five strains) were complemented by the CBF5
genes, respectively. Cbf5p and Nop58p are both components of snoRNP complexes. The large number of snoRNA species present in eukaryotic cells can be divided into two families on the basis of conserved sequence elements (reviewed in reference 41
). Nop58p associates with the box C+D family of snoRNAs (18
), most of which function as guides to direct ribose methylation on pre-rRNA (35
). Cbf5p is likely to be the rRNA pseudouridine synthase which is guided by the box H+ACA family of snoRNAs to sites of pseudouridine formation on pre-rRNA (17
). In addition to their roles in pre-rRNA modification, both classes of snoRNA include members that are critical for pre-rRNA processing at three early cleavage sites designated A0
, and A2
(see Fig. ).
In the presence of functional CBC, the two LUC8 strains and five LUC9 strains were temperature sensitive for growth at 37°C and the LUC9 strains were additionally strongly cold sensitive for growth at 16°C (Fig. ). Following transfer from 25 to 37°C, the luc8-sl1
strain showed an inhibition of pre-rRNA processing (Fig. and A), while the luc8-sl2
strain showed a largely nonconditional processing inhibition (Fig. A and C). The processing defects resemble those seen in strain depleted of Cbf5p; the 35S pre-rRNA accumulated, while the 32S, 27SA2
, and 20S pre-rRNAs were depleted (Fig. ). Aberrant processing intermediates (the 21S, 22S, and 23S RNAs) were also detected (Fig. A and data not shown). These phenotypes are indicative of the inhibition of processing at sites A0
, and A2
. The LUC9 strains showed a mild pre-rRNA processing defect at 37°C (Fig. A) and stronger inhibition of processing following transfer from 30 to 16°C (Fig. C). Again, the phenotype was indicative of the inhibition of processing at sites A0
, and A2
. Similar inhibition is seen in strains genetically depleted of Nop58p (80
FIG. 3 Growth of the LUC8 and LUC9 strains carrying functional CBC. Dilutions (1- to 102-fold) of luc8 and luc9 strains along with the wild-type isogenic (WT) control strain were spotted on minimal plates at 16, 23, 30, and 37°C and incubated for 3 days. (more ...)
FIG. 5 Northern analysis of pre-rRNA (A and C) and snoRNA (B and D) levels in LUC8 and LUC9 strains. RNA was extracted following growth at 23°C and 18 h after transfer to 37°C (A and B) or following growth at 30°C and 12 h after transfer (more ...)
The SL strains LUC8 and LUC9, expressing yCBP80 and yCBP20, have both rRNA processing and snoRNA stability defects that are consistent with mutations in CBF5
, respectively (Fig. ). Nop58p is required for the stability of the box C+D class of snoRNAs, while Cbf5p is required for stability of box H+ACA snoRNAs (18
). The luc8-sl1
strain was found to result in conditional depletion of the box H+ACA snoRNA snR3 at 37°C, while luc8-sl2
resulted in nonconditional depletion of snR3 (Fig. B and D). Depletion of the essential box H+ACA snoRNA, snR30, was substantially less marked at 23 or 30°C (data not shown). None of the LUC9 strains resulted in clear depletion of the box C+D snoRNA, U14 (Fig. B and D).
The genetic interaction of CBC with components of both major classes of snoRNP suggested that deletion of CBC might affect pre-rRNA processing. This possibility was tested by Northern hybridization using probes specific for either mature rRNAs (Fig. A) or pre-rRNAs (Fig. B to F) in strains lacking yCBP80 and/or yCBP20.
FIG. 6 yCBC is required for normal pre-rRNA processing. For Northern blot analysis of mature and precursor rRNAs, RNA was extracted from wild-type (WT) and cbp strains as indicated. (A) Hybridization with a probe complementary to the mature 18S and 25S RNAs; (more ...)
Several pre-rRNA species accumulated to abnormally high levels in all three deletion strains; the 35S primary transcript, the 32S pre-rRNA, and an aberrant 21S rRNA (see also Fig. ). In contrast, the level of the 27SA2 pre-rRNA was strongly reduced. The 21S intermediate extends from site A0 to A3, and results from cleavage of the 32S pre-rRNA at site A3 in the absence of cleavage at site A2. We conclude that A2 cleavage is particularly inhibited in the mutants. The overall pattern of defects, however, suggests that not only A2, but also the A0 and A1 cleavage events are slowed. Levels of 27SB and 7S pre-rRNAs were not altered (Fig. E and F), indicating that the pathway of 5.8S/25S rRNA synthesis is not affected by yCBC deletion (Fig. ). We conclude that the absence of Cbp20p or Cbp80p inhibits processing at sites A0, A1, and A2, with the greatest effects on A2. Processing at later steps on the pathway of 5.8S/25S synthesis does not appear to be affected. No clear reduction in the levels of mature 25S or 18S rRNAs was observed (Fig. A and G), so the inhibition of mature rRNA synthesis is unlikely to be directly responsible for the slow growth of the cbp deletion strains.
Four snoRNA species are required for pre-rRNA processing at sites A0
, and A2
: U3 and U14, which are associated with Nop58p (27
), and snR30 and snR10, which are associated with Cbf5p (40
). Among these, the rRNA processing phenotype observed in the cbp
deletions strains is most similar to this observed upon deletion of the SNR10
). Depletion of Nop58p or Cbf5p leads to loss of the snoRNAs with which they are associated; moreover, several snoRNAs, including U18 and U24, are encoded within pre-mRNA introns, and their synthesis could be inhibited by splicing defects. We therefore examined the steady-state levels of snoRNAs in the CBC deletion strains. No change in the steady-state levels of any of these snoRNAs was observed (Fig. and data not shown).
yCBC does not affect accumulation of various snoRNAs. For Northern blot analysis of low-molecular-weight RNAs, RNA was extracted from wild-type and cbp strains as indicated. The probes used for hybridization are described in Materials and Methods.
Many snoRNAs, including U3, snR10, and snR30 snoRNAs, carry hypermethylated 5′ cap structures (26
). The cap structures on the U3 and U8 snoRNAs have been reported to be required for nucleolar localization (28
; but see reference 42
) and therefore, presumably, for function. The efficiency of cap hypermethylation in the cbp
strains was assessed by immunoprecipitation using a m2,2,7
G cap-specific serum (R1131) and an monoclonal antibody that reacts with both m2,2,7
G and m7
G cap structures (H20; kindly provided by R. Lührmann). No difference in immunoprecipitation was observed between RNAs extracted from the wild-type and cbp20
-Δ strains, suggesting that the cbp
strains were not deficient in snoRNA cap hypermethylation (data not shown).
Defects in ribosome assembly caused by inefficient splicing of the pre-mRNAs encoding ribosomal proteins can inhibit pre-rRNA processing in yeast (references 8
and references therein). Since processing defects were detected mainly in the small ribosomal subunit rRNA, we first investigated the splicing of small subunit ribosomal protein (RPS
) pre-mRNAs in the CBC deletion strains. As a control, we utilized the temperature sensitive prp2-1
strain, which exhibits a strong splicing block at the nonpermissive temperature (37°C) and consequent accumulation of pre-mRNAs (reference 58
and Fig. ). Since CBC plays roles in the U1 snRNP-5′ splice site interaction and commitment complex assembly (10
), we initially analyzed pre-mRNAs with nonconsenus 5′ splice sites (73
GU instead of GUAU
GU; while RPS11A
instead of GUAUGU
FIG. 8 yCBC affects steady-state levels of mRNAs of ribosomal proteins. RNA was extracted from either wild-type or cbp strains as indicated. Additionally, control RNA was extracted from a temperature-sensitive-lethal splicing-deficient prp2-1 strain grown either (more ...)
Analysis of the steady-state levels of these mRNAs shows that splicing of the pre-mRNAs is inhibited. A similar degree of splicing inhibition was observed in the single and double cbp
deletion strains (Fig. A, I to VI), while the different pre-mRNAs showed various degrees of inhibition. The level of RPS11A
mRNA was not significantly altered, and there was no detectable accumulation of nonspliced pre-mRNA. By contrast, the mature RPS9A
, and RPS11B
mRNAs were depleted in the deletion strains and pre-mRNAs accumulated (Fig. A, I to VI; Table ). The accumulation of unspliced precursors indicated that splicing of these pre-mRNAs is indeed defective. The defects in the splicing of RPS9A
were particularly strong. These differences may be explained by examination of the pre-mRNA sequences. In addition to nonconsensus 5′ splice sites, RPS9A
also lack optimal polypyrimidine tract and branchpoint region sequences (C
ACUAAC and G
, respectively, instead of UACUAAC). Since reporter introns with either 5′ splice site or branchpoint mutations are very poorly spliced in strains lacking yCBC (references 10
), this may contribute to their inefficient splicing in the absence of CBC. The splicing of actin pre-mRNA (Fig. A, XVIII) was not altered in the yCBC deletion strains. RPS3
encodes a small subunit ribosomal protein but does not contain an intron (Fig. A VII), while the introns in RPS10A
(data shown only for RPS10A [Fig. A, VIII]) have a consensus 5′ splice site. For each of these genes there was a clear decrease in mRNA, although this was not accompanied by an increase in the RPS10A
pre-mRNAs (data not shown).
TABLE 2 PhosphorImager (Molecular Dynamics) analysis of the accumulation of RPS9A, RPS11B, RPL30, and RPL28 pre-mRNAs andmRNAsa
Since the levels of many pre-mRNAs and mRNAs encoding small subunit ribosomal proteins were affected in cbc strains, we examined whether this would also be the case for large ribosomal subunit (RPL) pre-mRNAs. The 5′ splice sites of these pre-mRNAs are GUACGU for RPL16A and RPL16B, GUAUGA for RPL22A, GUACGU for RPL22B, and GUCAGU for RPL30, compared to the consensus GUAUGU (Fig. B). The steady-state levels of these mRNAs were also decreased in all cbc strains, particularly for RPL16A and RPL16B (Fig. A, IX-XIV; RPL16B and RPL22B were very similar to RPL16A and RPL22A [data not shown]). The decrease in the level of mRNA was clearly accompanied by accumulation of pre-mRNA only in the case of RPL30 (Table ). Note that the 5′ splice site of RPL30 pre-mRNA has two nonconsensus residues. RPL25 and RPL28 (CYH2) pre-mRNAs have consensus 5′ splice sites, whereas RPL10 pre-mRNA has no intron. There was no significant reduction in the levels of these three mRNAs in the cbc strain. We conclude that the splicing of the pre-mRNAs with suboptimal splice sites is strongly inhibited in strains lacking CBC. The degree of inhibition varies between different pre-mRNAs with weak 5′ splice sites (Fig. A; Table ), reflecting the differing relative levels of importance of CBC function in the splicing of those pre-mRNAs. Note that the reduction in some ribosomal protein mRNAs without concomitant increase in pre-mRNA, and the reduction in mRNAs from genes without introns, could well be a consequence of the impaired growth of the strains and consequent reduction in ribosome synthesis.
Other SL complementing genes.
LUC11 (Table ) is complemented by GCR1
, a transcriptional activator required for expression of multiple genes involved in glucose metabolism (3
). Since the gene encoding yCBP80, GCR3
, was first identified in a search for additional mutants that affected growth on glucose (75
), the finding of gcr1
mutants in our screen was not a surprise. As would be expected, the alleles of GCR1
recovered from the synthetic lethal screen did not cause lethality when the strains were plated on nonfermentable carbon sources (data not shown). Three additional complementation groups among the seven for which no complementing plasmid was recovered also failed to produce lethality when grown on nonfermentable carbon sources, suggesting that additional genes involved in glucose metabolism are likely to be involved in producing the SL phenotype. A possible explanation for both the earlier and present findings with GCR1
is the report that the GCR1
gene includes an intron with a nonconsensus 5′ splice site (GUAUGA
instead of GUAUGU
There is no obvious reason why SRV2
(LUC12) or any of the genes on the LUC14 complementing plasmid (Table ) should, when mutated, generate a lethal phenotype in the absence of CBC. Similarly, although it is possible to speculate on possible functional connections between pol III transcripts (e.g., U6 snRNA) and CBC, the identity of any of the genes that complement LUC13 is not readily explicable. Given the role of CBC in U snRNA transport in vertebrates (31
) and the existence of an abundant complex in yeast between CBC and yeast importin α (Srp1p), a mediator of nuclear protein import (20
), it was of interest that the temperature-sensitive allele of LUC10/SSD1
recovered in this screen accumulates poly(A)-containing RNA in the nucleus at nonpermissive temperature (data not shown). LUC10/SSD1
was the only strain isolated in the screen showing this phenotype. However, an ssd1
-Δ allele with a precise deletion of the entire ORF did not exhibit nuclear poly(A) accumulation (21a
). Sequence analysis suggests that SSD1
likely encodes an exonuclease of the RNase II family (76
), consistent with a role in RNA metabolism, but no change in mRNA stability was detected in either the cbp
deletion strains or luc10-1