RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

Abstract

Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30°C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.

The synthesis of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I is an important step in the synthesis of ribosomes, and its regulation is closely linked to the nutrient conditions and growth potential for the cell. Previous studies have identified essential components of the Pol I transcription apparatus as well as important cis-elements in Pol I promoters and revealed some potential mechanisms for regulation of transcription initiation (for reviews, see refs. 1–5). Unlike Pol II, however, little is known regarding mechanisms that regulate postinitiation steps of transcription by Pol I.

One well characterized Pol II transcription elongation factor in yeast is a complex of two proteins, Spt4p and Spt5p (the complex will be referred to as Spt4/5 here). The SPT4 and SPT5 genes were among many genes isolated for their ability to suppress transcription defects caused by insertions of the retrotransposon Ty1 (or the Ty1 long terminal repeats, δ) in the 5′ noncoding regions of yeast genes (6). Swanson and Winston (7) later showed that Spt4/5 associates with Spt6p, which affects Pol II elongation through chromatin. However, Spt4p and Spt5p also form a separate complex, devoid of Spt6p, that associates with Pol II physically and genetically, and this interaction is important for transcription elongation (8). More recent work has shown that deletion of the nonessential gene SPT4 results in reduced efficiency of Pol II elongation through GC-rich DNA sequences (9) and a general decrease in Pol II processivity (10). Taken together, all of these data clearly support a role for Spt4/5 in transcription elongation by Pol II in yeast.

Spt4/5 also plays a role in Pol II transcription elongation in mammalian cells. The mammalian homologues of Spt4p and Spt5p form a complex called the 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF), which was originally identified as a factor that induces a DRB-dependent arrest of elongating Pol II complexes in reconstituted in vitro transcription assays (11). It was demonstrated that under nucleotide-limiting conditions, DSIF could also increase the rate of elongation of Pol II in vitro. Thus, work in mammalian cells suggests a role for DSIF (Spt4/5) in transcription elongation, but the exact nature of its effect may vary depending on various regulatory sequence elements in a given transcription unit. In fact, there is strong evidence indicating that transcription by Pol II is coupled to modification of its mRNA product, and this coupling involves the activity of Spt4/5. After initiation of transcription by Pol II, there is a promoter-proximal pause that is mediated by DSIF (Spt4/5) and the negative elongation factor. This pause presumably acts as a checkpoint to permit efficient capping of the mRNA before continued elongation. Once the mRNA is properly capped, a cyclin-dependent protein kinase, P-TEFb (mammalian homologue of yeast Ctk1p), phosphorylates the Spt5p subunit of DSIF, relieving the pause and promoting elongation (for a review see ref. 12).

In addition to their defined roles in cellular Pol II activity, Spt5p is also involved in Tat-activated transcription elongation during HIV infection (13), and Spt4p is required for efficient epigenetic silencing of transcription and chromosome segregation in yeast (14, 15). Here, we describe evidence indicating that Spt4p, presumably as an Spt4/5 complex, is involved in elongation steps of rRNA transcription by Pol I, perhaps playing a role in coupling rRNA processing to transcription.

Results

Spt4p and Spt5p Associate with RNA Polymerase I.

To identify factors that contribute to regulation of Pol I activity, we purified the polymerase and analyzed these preparations using mass spectrometry. We used a three-step method for purification of Pol I from a strain that contains an N-terminal tandem epitope tag, (His)₆-hemagglutinin (HA)₃, on the second largest Pol I subunit, A135 (see Materials and Methods). We have analyzed several Pol I preparations using mass spectrometry and detected copurification of the Pol II elongation factors Spt4p and Spt5p with Pol I (Table 1). Because we did not detect copurification of Pol II subunits, it seemed likely that Spt4/5 was associated with Pol I. Upon inspection of the literature, we found that a previous analysis of proteins that associated with Spt5p identified several subunits of Pol I in addition to the expected Pol II subunits and other factors (16), supporting our results.

Table 1.

Selected mass spectrometry results of polymerase preparation from NOY760

Protein	Molecular mass, kD	No. of peptides	No. of spectra	Coverage, %
A12	13.6	34	308	88.8
A135	135.7	307	5,445	86.1
Spt4p	11.2	9	75	88.2
Spt5p	115.6	35	133	29.4
Rrn3p	72.4	18	81	39.9

MS results from a representative polymerase preparation from NOY760 grown in YEPD to an A₆₀₀ of ≈0.6. Summary of detection data for two representative polymerase subunits (A12 and A135), Spt4p, Spt5p, and a Pol I-associated transcription factor, Rrn3p, are shown.

To confirm the association of Spt5p with Pol I, we used antibodies to the largest Pol I subunit (A190) to immunoprecipitate Pol I in crude extracts made from strains containing either (His)₇-(HA)₃-tagged Spt5p or (His)₆-HA-tagged Rrn3p, which is known to associate with Pol I during initiation of transcription (17–19) and was used as a positive control. We then analyzed the immunoprecipitated (IP) fractions by Western blot using anti-HA antibodies and observed a clear coprecipitation of Spt5p, as well as Rrn3p, with Pol I (Fig. 1). These data support the results observed by using mass spectrometry and prompted us to further examine potential roles for Spt4/5 in Pol I transcription.

Fig. 1.

Spt4/5 associates with Pol I. Pol I was immunoprecipitated from equivalent amounts of crude extracts made from NOY761 (HA-Rrn3p) and NOY2164 (HA-Spt5p) by using a polyclonal antibody against A190. For control, identical extracts were treated with protein A-Sepharose beads only. Coprecipitated HA-tagged Rrn3p and Spt5p were detected by Western blot using the monoclonal antibody 12CA5.

Spt4p and Spt5p Are Associated with rDNA.

If the association of Spt4/5 with Pol I has functional significance in Pol I transcription elongation, as in Pol II, then both proteins are expected to be associated with Pol I when the polymerase is engaged in transcription of the rDNA. To test this hypothesis, we measured the association of Spt4p and Spt5p with the rDNA in exponentially growing cells using ChIP assays. We used three strains; each contained an identical epitope tag, (His)₇-(HA)₃, on the C terminus of Spt4p, Spt5p, or the Pol I subunit A135 (as a positive control). We found that both Spt4p and Spt5p associate with rDNA in vivo, and they associate, relative to control Pol I association, preferentially with the transcribed region of the rDNA rather than with the promoter (Fig. 2). These data are consistent with the model that Spt4/5 functions in transcription elongation by Pol I.

Fig. 2.

Spt4p and Spt5p associate with rDNA in vivo. ChIP analysis of (His)₇-(HA)₃-tagged Spt4p, Spt5p, and A135 (Pol I) was performed by using strains NOY2165, NOY2164, and NOY2166, respectively. NOY388 was also analyzed as “no tag” control. Anti-HA antibody 12CA5 was used for IP, and two dilutions of both input and IP DNA were analyzed by PCR to verify that the data were in the linear range of detection. Two regions of the rDNA were examined: the promoter region from −251 to −24 and the 25S coding region from +3586 to +3876. Numbering is relative to the initiation site of Pol I. ChIP data from three independent cultures were quantified by using a PhosporImager, and the ratios of IP/input were averaged for each region of the rDNA and were normalized to the values for Pol I.

The degree of association of Spt4p or Spt5p with the rDNA was less than that of Pol I (Fig. 2). The most likely explanation for this observation is that Spt4/5 associates with Pol I rather than with the rDNA directly, similar to the association of Spt4/5 with Pol II (8, 15). If this explanation is true, then one would expect the efficiency of precipitation of DNA through a network of protein–protein interactions to be lower. However, we cannot exclude the possibility that Spt4/5 associates directly with the rDNA, but with a lower affinity or stoichiometry than that of Pol I.

Effects of Deletion of SPT4 on rRNA Transcription.

In yeast, SPT5 is an essential gene; however, SPT4 can be deleted. Previous studies on the roles of Spt4/5 in Pol II transcription have used spt4Δ strains, which disrupt the function of the Spt4/5 complex (at least partially) (14, 15, 20). To test for a potential role for Spt4/5 in Pol I transcription, we replaced the SPT4 gene with the HIS3MX6 gene (21). This spt4Δ strain exhibited temperature-sensitive growth at 37°C as reported (14) and grew somewhat more slowly (≈20%) than the WT parental strain at permissive 30°C.

The total RNA synthesis rate was initially measured by pulse-labeling cells for 2.5–5 min with [methyl-³H]methionine. The spt4Δ mutant cells showed only a small decrease (10–20%) in the incorporation of the ³H-label into the RNA fraction relative to WT control cells (data not shown; for the analysis of individual rRNA species, see below). We also carried out [³H]uridine pulse-labeling experiments. We found that, although incorporation into the RNA fraction decreased ≈2-fold in the mutant, the specific activities of [³H]UTP during pulse-labeling was lower in the spt4Δ strain than the control strain. Overall, the synthesis rate of total RNA was calculated to be lower in the mutant by ≈31% than in the WT (per cell mass; Table 2). Analyses of ³H-labeled RNA by gel electrophoresis showed that the degree of the decrease in labeling was approximately the same for all of the stable RNAs, rRNAs, 5S RNA, and tRNA (data not shown), indicating that the rate of rRNA synthesis was also decreased to the same extent as total RNA (≈31%).

Table 2.

Relative rates of RNA synthesis in the spt4Δ and WT strains

Strain	[3H]uridine incorporation (a)	Sp. act. of UTP (b)	Synthesis rate (a)/(b)
WT	1.0	1.0	1.0
spt4Δ	0.45 ± 0.05	0.65 ± 0.06	0.69 ± 0.10

NOY388 (WT) and NOY2167 (spt4Δ), each carrying pRS316 (to make cells Ura⁺), were grown in SD-Ura at 30°C to A₆₀₀ = ≈0.3. Cells were pulse-labeled with [³H]uridine for 5 min, and incorporation of [³H]uridine into TCA-precipitable total RNA fraction was measured. Three independent cultures were used for each strain, and the average value was calculated for the rate of incorporation in the spt4Δ relative to WT strains. Under the same experimental conditions, specific activities (Sp. act.) of ³H-labeled UTP were measured for each strain by using steady-state labeling of pool UTP with [³²P]- and [³H]uridine pulse-labeling for 2.5, 5, and 10 min (see Supporting Materials and Methods). Sp. act. were approximately the same at these three time points for each of the strains. Averages of the values for 2.5 and 5 min were used as average Sp. act. of UTP during the 5-min pulse-labeling, and the data from two independent experiments were used to calculate the relative Sp. act. shown in the table. The relative total RNA synthesis rate in the spt4Δ was then calculated from the incorporation data corrected for the difference in pool UTP Sp. act. between the two strains.

The spt4Δ Mutation Affects the Elongation Step in rRNA Transcription.

Although deletion of SPT4 decreased growth rate and rRNA synthesis rate only weakly, previous studies indicated stronger effects on transcription of some Pol II genes as mentioned above. In particular, using an artificial fusion gene, a long (≈8-kb) uncharacterized ORF fused to the GAL1 promoter, Mason and Struhl (10) concluded that deletion of SPT4 caused a ≈2-fold decrease in processivity but did not affect the elongation rate. Thus, we asked whether similar processivity defects might be observed for Pol I transcription of rRNA genes in the spt4Δ mutant. The processivity assay used by Mason and Struhl (10) compares the association of Pol II with the 5′ end of the gene to that at the 3′ end using ChIP analysis. This method is assumed to measure the processivity of the polymerase, or its ability to stay engaged on a template from initiation to termination of transcription. When we used this assay for rRNA transcription with the spt4Δ and WT strains, we observed a reproducible decrease in the apparent processivity of Pol I in the mutant strain compared with WT (Fig. 3). The 2-fold decrease in processivity of Pol I for the ≈6-kb transcribed region that we observed in the spt4Δ strain is nearly identical to the published defect in processivity of Pol II for an ≈8-kb transcribed region in the spt4Δ mutant (10). These data suggest that deletion of SPT4 may result in reduced efficiency of Pol I elongation through rDNA, possibly by reducing processivity of the enzyme, as was concluded for its effects on Pol II transcription.

Fig. 3.

Processivity analysis of Pol I transcription in the spt4Δ strain relative to WT. NOY388 (WT) and NOY2167 (spt4Δ) (both carrying pRS316) were grown to A₆₀₀ = ≈0.3 in SD-Ura medium. (A) Pol I association with the 5′ end (+67 to +388) and the 3′ end (+6067 to +6388) of the 35S rRNA coding region was then measured by ChIP using anti-A190 antibodies. Two dilutions of input and IP DNA were measured by PCR. As a negative control, duplicate samples were treated with protein A-Sepharose beads only (“−Ab”) and diluted similarly to IP samples. (B) For each strain, the IP/input value for the 3′ end was divided by the value for the 5′ end, and the ratio (“processivity”) for the mutant was normalized to that for WT. The values obtained from four independent IPs from two independent cultures were averaged. Raw values for IP/input were 0.060 ± 0.014 for the 5′ end of WT, 0.013 ± 0.002 for the 3′ end of WT, 0.124 ± 0.004 for the 5′ end of spt4Δ, and 0.017 ± 0.003 for the 3′ end of spt4Δ.

EM Analysis of Effects of the spt4Δ Mutation on Pol I Transcription and rRNA Processing.

We examined rRNA synthesis in both the spt4Δ and WT strains by EM Miller chromatin spread analysis and observed multiple different effects of the spt4Δ mutation on rRNA synthesis.

Contrary to the simple prediction from the ChIP processivity assay described above (Fig. 3), we observed no obvious difference in the density of Pol I molecules near the 3′ end relative to the 5′ end of individual 35S rRNA genes between the mutant and WT strains (Fig. 4A and other EMs not shown). If the loss of Spt4p function resulted in a simple 2-fold decrease in Pol I processivity, EM analysis of Miller spreads would detect such a defect. Thus, Spt4p (perhaps together with Spt5p) seems to affect Pol I transcription in a more complex way(s) rather than simply decreasing processivity of elongation.

Fig. 4.

Representative EMs of rRNA genes from the spt4Δ strain relative to WT. (A) NOY388 (WT) and NOY2167 (spt4Δ) were grown in YEPD plus 1 M sorbitol to midlog phase. Miller spreads were analyzed by EM, and two active genes are shown for each strain. (Scale bars, 0.5 μm.) (B) The number of Pol I molecules per gene was counted, and distribution of Pol I among genes is plotted by grouping with an increment of 5. (C) Numerical values for the data presented in B and the active rDNA repeats (as percentage of total) counted for contiguously traced rDNA regions (23) are shown. The values for the mutant normalized to those for the WT (100) are also shown in parentheses. N, the number of genes examined per strain; pols, polymerases.

We quantified the Pol I density per rDNA gene in the WT and spt4Δ strains. We observed an overall increase in polymerase density per gene in the spt4Δ mutant compared with WT (Fig. 4 A–C). Most noticeably, there is a significant reduction in the number of genes with lower polymerase density (Fig. 4B). Calculation showed that the average number of polymerases per gene in the spt4Δ strain was ≈30% higher than in WT (Fig. 4C). Thus, we found that an ≈31% reduction in the rRNA synthesis rate in the spt4Δ strain (Table 2) is accompanied by an ≈30% increase in the number of polymerases per gene compared with WT.

In WT yeast cells, only a fraction of the rRNA genes are actively transcribed (22, 23) The fraction of active versus inactive rDNA genes can be quantified by EM. Using the method described in ref. 23, we found a small (18%) increase in the percentage of active rDNA genes in the spt4Δ mutant compared with WT (Fig. 4C). However, we found that the mutant cells exhibit a large (≈3-fold) reduction in total rDNA copy number per genome (Fig. 5). The reason for this decrease, although interesting, has not been studied. From all of these data, one can calculate that the number of Pol I molecules engaged in rRNA transcription in the spt4Δ mutant relative to WT strains is ≈55% (1.29 × 1.18 × 0.36 = 0.55). This value, although it is subject to cumulative errors from measurements, is slightly lower than, and perhaps not significantly different from, the value (69%) obtained for rRNA synthesis rate in the spt4Δ mutant relative to WT (Table 2). Therefore, even though the spt4Δ mutation clearly alters the transcription pattern of individual active rRNA genes, the overall change in the elongation rate (or efficiency/processivity) of Pol I, relative to that in the control strain, (0.69/0.55 = 1.25) is small and perhaps not significant.

Fig. 5.

rDNA copy number is reduced in the spt4Δ strain compared with WT. (A) genomic DNA was extracted from three independent isolates of spt4Δ (NOY2167) and three independent WT cultures (NOY388) as well as from cultures with known rDNA copy numbers: NOY1071 (25 copies), NOY886 (42 copies), and NOY1064 (143 copies). After digestion with HindIII, DNA was separated in a 1.5% agarose gel and transferred to a nylon membrane. Southern hybridization was performed using ³²P-labeled DNA probes against rDNA and the single copy control gene URA3. To visualize the URA3 probe clearly (Lower), exposure time was extended. (B) Data from A were quantified by using a PhosphorImager, and the ratio of rDNA/URA3 of the reference strains was plotted against copy number. From the resulting linear regression, the average copy number in WT and spt4Δ was calculated. Variations (standard deviations) among three independent cultures were ≈15% for both WT and spt4Δ.

The spt4Δ Mutation Causes Defects in rRNA Processing.

Earlier studies showed that small 5′ terminal knobs seen on transcripts of all eukaryotic rRNAs by EM contain U3 small nucleolar RNA (snoRNA) and other proteins required for rRNA processing and 18S rRNA maturation (24, 25). Subsequent studies in yeast showed that the small terminal knobs become a large particle [called SSU (small subunit) processome] concomitant with compaction of pre-18S rRNA. The SSU processome is often lost by cotranscriptional cleavage, although the efficiency of this cleavage is not 100% (26). We examined rRNA processing events by EM in the spt4Δ and WT strains. We observed a substantial (≈2-fold) reduction in the efficiency of SSU processome formation and cotranscriptional rRNA cleavage in the spt4Δ strain compared with WT (Fig. 6 A and B).

Fig. 6.

Processing of rRNA transcripts is impaired in the spt4Δ strain. (A) One of the two genes shown in Fig. 4A for NOY388 (WT) and for NOY2167 (spt4Δ) is shown in higher magnification with notes indicating the absence of cotranscriptional cleavage of the 35S rRNA in the gene from the spt4Δ strain. (Scale bars, 0.5 μm.) (B) Quantification of SSU processome formation and cotranscriptional cleavage of rRNA observed by EM in the WT and spt4Δ strains, classified as efficient, intermediate, or slow (26). (C) The two strains were grown in SD (−Met) media and were pulse-labeled for 2.5 and 5 min with [methyl-³H]methionine. RNA was isolated and analyzed by using formaldehyde-agarose gel electrophoresis, followed by autoradiography. Positions of rRNA species are indicated. Two exposures of the blot are shown [1 day (Left) and 1 week (Right)]. The ratios of precursors to mature products detected after 2.5-min pulse-labeling are indicated, normalized to WT. (D) Analysis of polysomes and ribosomal subunits in the two strains. Cells were grown in YEPD medium at 30°C, and extracts were analyzed by sucrose density gradient centrifugation. Positions of 40S, 60S, and 80S as well as polysomes are indicated. The arrows with an H indicate halfmers in the spt4Δ strain.

The defects in rRNA processing were also observed in [methyl-³H]methionine pulse-labeling experiments. We clearly observed increased ³H-labeling of the 35S rRNA precursor and modestly increased labeling of 27S and 20S precursors relative to labeling of mature rRNAs after 2.5-min labeling in the spt4Δ strain compared with WT (Fig. 6C). A similar increase in labeled 35S pre-rRNA in the spt4Δ relative to WT strains was also observed in 5-min [³H]uridine pulse-labeling experiments (data not shown). These observations are consistent with the EM data, supporting the conclusion that disruption of SPT4 results in impaired cotranscriptional processing of rRNA.

We further tested whether the spt4Δ mutation affected ribosome assembly by analyzing polysome profiles in the mutant strain compared with the WT. As shown in Fig. 6D, the spt4Δ strain showed a small but reproducible reduction in the amount of free 60S subunits and an appearance of “halfmers” which represent polyribosomes containing an additional 40S subunit (27), a characteristic indicating preferential reduction in 60S relative to 40S subunit production (28) or a defect in 60S function (due to improper modification or assembly) (29).

The spt4Δ mutant synthesizes rRNA at a modestly reduced rate and shows reduction in 60S relative to 40S production as well as defects in rRNA processing. To confirm our hypothesis that the defects in rRNA processing are caused by improper Pol I elongation and not defects in Pol I initiation or as an indirect consequence of slow growth, we repeated the experiments using an rrn3-ts mutant (NOY1075, Table 3) at a semipermissive temperature (30°C). The rrn3 mutation specifically impairs transcription initiation (17, 30). At 30°C, this mutant showed both growth and rRNA synthesis rates slightly lower than those exhibited by the spt4Δ mutant. Nevertheless, the mutant did not show any defects in rRNA processing as analyzed by [methyl-³H]methionine labeling nor any abnormal pattern of polysomes and ribosomal subunits in sucrose-gradient analysis (data not shown). Taken together, these data strongly suggest that Spt4p (presumably as Spt4/5) plays a role in rRNA processing and ribosome assembly, most likely by affecting Pol I elongation.

Table 3.

Yeast strains and plasmids used in this study

Strain or plasmid	Description
Strains
NOY388	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100
NOY760	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 rpa135Δ::LEU2 carrying pNOY442
NOY761	MATα ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 rrn3Δ::HIS3 carrying pNOY685
NOY886	MATα ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 rpa135Δ::LEU2 fob1Δ::HIS3, carries     pNOY117, rDNA copy number ∼42
NOY1064	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 fob1Δ::HIS3 rDNA copy number ∼143
NOY1071	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 fob1Δ::HIS3 rDNA copy number ∼25
NOY1075	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 rrn3(S213P)
NOY2164	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 SPT5-(HA)3-(His)7:HIS3MX6
NOY2165	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 SPT4-(HA)3-(His)7:HIS3MX6
NOY2166	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 RPA135-(HA)3-(His)7:HIS3MX6
NOY2167	MATa ade2-101 ura3-1 trp1-1 leu2-3,112 his3-11 can1-100 spt4Δ::HIS3MX6
Plasmids
pRS316	pBluescript, CEN6, ARSH4, URA3 see (45)
pNOY117	pRS314 (pBluescript, CEN6, ARSH4, TRP1) derivative carrying RPA135
pNOY442	pRS314 (pBluescript, CEN6, ARSH4, TRP1) derivative carrying (His)6-(HA)3-RPA135
pNOY685	pRS315 (pBluescript, CEN6, ARSH4, LEU2) derivative carrying (His)6-(HA)-RRN3

Discussion

The data presented here demonstrate that both Spt4p and Spt5p physically associate with Pol I, presumably as a complex (Spt4/5); they also associate with rDNA, preferentially in the transcribed region. Deletion of SPT4 weakly reduces the rRNA synthesis rate and alters the pattern of distribution of elongating Pol I on rRNA genes. Pol I elongation is affected by the spt4Δ mutation as judged by the results of ChIP processivity assay, and the spt4Δ mutation leads to defects in rRNA processing, presumably as a result of improper elongation, as further discussed below. Based on these data, we conclude that this conserved Pol II elongation factor plays an important role in transcription elongation by Pol I as well. Furthermore, it is possible that the human homologue of Spt4/5, DSIF, also plays a role in Pol I transcription of rRNA genes.

Two other factors that affect elongation by Pol II have been recently implicated in transcription by Pol I. The phosphatase Fcp1p dephosphorylates the C-terminal domain (CTD) of Pol II and is generally required for Pol II transcription in yeast (31, 32). It was reported that fcp1-ts mutants had defects in Pol I transcription, and Fcp1p stimulated transcription by Pol I in vitro in specific and nonspecific transcription assays, supporting a role for Fcp1p in Pol I elongation (33). Another protein with well defined roles in elongation by Pol II is CTD kinase I (CTDK-1). The catalytic subunit of CTDK-1, Ctk1p (or the mammalian homologue, P-TEFb), phosphorylates the Ser-2 position of the CTD of Pol II as well as Spt5p to clear a promoter-proximal checkpoint that ensures proper capping of the mRNA (for a review see ref. 12). Bouchoux et al. (34) have shown that CTDK-1 associates with Pol I, and deletion of CTK1 results in impaired Pol I transcription, suggesting that CTDK-1 plays a role in Pol I transcription. These recent works, together with our identification of a role for Spt4/5 in Pol I transcription and studies on both TFIIS and TFIIH (35, 36), demonstrate an increasing degree of similarity between transcription elongation by Pol I and Pol II.

Coupling of rRNA Processing to Transcription by Pol I.

We showed that disruption of SPT4 results in defects in rRNA processing and ribosome assembly. It is important to note that we cannot exclude indirect effects of the deletion of SPT4 on rRNA processing (via pathways that do not involve Pol I), especially because Spt4p and Spt5p have known roles in Pol II transcription. However, given that Spt4/5 physically associates with Pol I and rDNA, the simplest explanation is that loss of Spt4p function impairs Pol I elongation, leading to defects in regulated cotranscriptional processing of the rRNA transcript. In support of this conclusion, we have recently demonstrated that specific mutations in Pol I itself impair elongation, rRNA processing, and ribosome assembly (our unpublished experiments).

Based on existing data and similarities to the Pol II system, Ctk1p might modulate the activity of Spt5p to regulate cotranscriptional rRNA processing events during transcription of rRNA by Pol I; however, it is unlikely that this is the only set of elongation factors that regulate Pol I transcription elongation and cotranscriptional rRNA processing.

A different type of coupling of Pol I transcription to ribosome assembly has recently been reported by Gallagher et al. (37). A subset of the proteins that comprise the SSU processome (called t-Utps) have dual roles, both in enhancing transcription by Pol I (apparently at the initiation) and in SSU assembly. When these proteins were depleted, there were substantial effects not only on rRNA processing but also on transcription by Pol I (37). Clearly, further studies are required to understand the mechanisms that coordinate synthesis of rRNAs and their processing and assembly into final ribosome structures.

Defining the Role of Spt4/5 in Pol I Transcription.

Spt4/5 has been shown to act both positively and negatively on elongation by Pol II (8, 9, 11, 13). The results of our ChIP analysis formally suggest that the processivity of Pol I is decreased ≈2-fold by the spt4Δ mutation (Fig. 3). However, examination of the EM pictures did not reveal a significant difference in polymerase densities at the 3′ end relative to the 5′ end of the rDNA genes between the mutant strain and the WT strain; i.e., no evidence for defects in Pol I processivity in the spt4Δ mutant was recognized (Figs. 4 and 6, and data not shown). Thus, there is an apparent discrepancy between the EM analysis and the ChIP analysis, and this discrepancy may have implications on the mechanism by which Spt4p might affect Pol I function.

One likely explanation for this discrepancy takes into account the possibility that ChIP analysis of complex enzymes that must engage the DNA in a variety of ways (e.g., polymerases, which initiate, pause, arrest, elongate, and terminate transcription) is not a simple assay for association or lack of association. For example, the accumulation of long nascent transcripts on transcribing polymerases might sterically inhibit IP with antibodies against polymerase subunits or decrease efficiency of cross-linking of Pol I to DNA. Similarly, various small nucleolar (sno)RNPs or protein factors recruited to paused or elongating polymerases might influence efficiencies of the cross-linking and/or immunoprecipitability. Thus, the reduced “association” of Pol I at the 3′ end of the rDNA relative to the 5′ end in the mutant strain compared with the WT strain may reflect an alteration in the mode of association of Pol I with the rDNA gene as a result of the loss of Spt4p function, or it might simply reflect a different degree or mode of association of various factors with elongating and/or paused Pol I caused by the spt4Δ mutation at these two regions selected for the ChIP analysis. In addition, we do not know why the synthesis of 60S subunits is apparently more sensitive to the spt4Δ mutation than 40S subunit synthesis or whether this is related to the observed difference in the ChIP analysis between the mutant and WT strains. These questions are subjects for future studies.

Previous work using this ChIP processivity assay suggested that the role of Spt4/5 in Pol II transcription is to increase processivity (10). We note that the ability to measure polymerase density by using Miller spreads is unique to Pol I (i.e., has not easily been done with Pol II), so the discrepancy between ChIP and EM results described here might also apply to some Pol II transcription units. Irrespective of the exact mechanistic steps of Pol I transcription affected by the spt4Δ mutation, the results obtained by using a variety of experimental approaches described in this article demonstrate that disruption of SPT4 causes defects in Pol I transcription, almost certainly during the elongation step, and leads to defects in rRNA processing and ribosome assembly.

Materials and Methods

Yeast Strains and Growth Conditions.

Yeast cells were grown at 30°C with aeration. Yeast extract/peptone/dextrose (YEPD) and synthetic glucose complete (SD) media were described (30, 38). Yeast strains and plasmids are listed in Table 3. SPT4 and SPT5 were tagged with a tandem epitope tag (His)₇-(HA)₃ at the C terminus of the encoded proteins or disrupted (spt4Δ) as described (21).

Protein Identification by Mass Spectometry.

Pol I was purified by an immunoaffinity method essentially as described in ref. 39, followed by an additional heparin Sepharose step as described in ref. 18. Methods used for mass spectrometry are described in Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Biochemical Analyses.

Coimmunoprecipitation of HA-tagged Spt5p and Rrn3p with Pol I was carried out by using extracts prepared from NOY2164 and NOY761, respectively. Extracts were incubated with polyclonal anti-A190 antibodies, followed by the addition of protein A-Sepharose beads. The beads were recovered, and coprecipitated HA-tagged proteins were detected by SDS/PAGE, followed by Western immunoblot analysis using a monoclonal anti-HA antibody (12CA5). More details are described in Supporting Materials and Methods.

Analysis of rRNA synthesis by labeling RNA with [³H]uridine or [methyl-³H]methionine was carried out as described (30, 40). To calculate total RNA synthesis rates from the observed rates of [³H]uridine incorporation, specific activities of [³H]UTP in an acid-soluble UTP pool were measured by the method adapted from ref. 41. Details are described in Supporting Materials and Methods.

ChIP analysis was performed essentially as described (10, 30, 42), with minor modifications that are outlined in Supporting Materials and Methods.

Quantification of rDNA copy number was performed as described (43), except that Southern blot hybridization of the rDNA probe was normalized to the URA3 gene probe. Sucrose gradient centrifugation analysis of polysome profiles was performed essentially as described (44).

EM Miller Chromatin Spreads Analysis.

Cells were grown in YEPD medium containing 1 M sorbitol. Miller chromatin spreads and analysis by EM were performed as described (23). The polymerase number per gene was quantified by counting all RNA polymerases or nascent transcripts on all rRNA genes that could be unambiguously traced from 5′ end to 3′ end.

Abbreviations

CTD

C-terminal domain

CTDK

CTD kinase

DSIF

5,6-dichloro-1-β-d-ribofuranosylbenzimidazole sensitivity-inducing factor

HA

hemagglutinin

IP

immunoprecipitated/immunoprecipitation

Pol

Polymerase

SSU

small subunit