Isolation of an rpa135 Mutant with Defects in Transcription Elongation
It was previously shown that 6-azauracil (6AU) inhibits growth of yeast cells by reducing cellular UTP and GTP levels (Exinger and LaCroute, 1992
), and that decreased nucleotide concentrations increase RNA polymerase pausing and elongation arrest in vitro (Uptain et al., 1997
). 6AU-hypersensitive (6AUS
) yeast mutants with alterations in Pol II subunits were also isolated that show defects in Pol II elongation in vivo (Archambault et al., 1992
; Langelier et al., 2005
). To isolate elongation-defective mutations in Pol I, we introduced PCR-mutagenized copies of RPA135
(gene for second largest Pol I subunit) to a yeast strain carrying an rpa135Δ
mutation (NOY975), which grows by virtue of Pol II transcription of the 35S rRNA coding region fused to the GAL7
-35S rDNA”) on a plasmid (see ). Several independent 6AUS
mutants (as judged by their growth on glucose with and without 6AU; cf. ) were isolated. One of these mutants contained a missense mutation resulting in a glycine at amino acid position 784 rather than an aspartate. The acidic residue at this position is conserved among most or all known multisubunit RNA polymerases, including yeast Pol I, yeast Pol II, human Pol II, and E. coli
RNA polymerase (). In Pol II this residue is located close to the catalytic center and might play a role in loading substrate NTP at the active site during Pol II transcription (Cramer et al., 2001
; Langelier et al., 2005
Yeast Strains and Plasmids Used in This Study
Mutation of Aspartate 784 to Glycine in A135 Impairs Pol I Elongation Rate
In Vitro Analyses of Pol I Carrying the rpa135(D784G) Mutation
We constructed strains expressing His6-(HA)3-tagged versions of WT (NOY2173) and mutant (NOY2174) A135. We purified Pol I from these strains and observed no difference in Pol I subunit composition or stability (as assessed by SDS-PAGE, data not shown).
Using a standard multiround Pol I transcription assay system with all purified initiation factors (Keener et al., 1998
), we found that when one of four NTP substrates was reduced (UTP and ATP analyzed), transcription by the mutant Pol I was reduced relative to the WT Pol I, and that the difference in the activities between the two polymerases was less at higher NTP concentrations (). This observation is consistent with the selected phenotype of the mutant, i.e., 6AU sensitivity. However, since these multiround assays do not differentiate defects in elongation from other kinetic steps (i.e., initiation, pausing, arrest, or termination), we developed an assay for Pol I elongation rate.
We constructed a linear rDNA template (−247 to +763) in which all 6 CG base pairs encoding C between +1 to +56 are replaced by GC base pairs (eliminating CTP requirement for transcription up to +56) to compare elongation rates in vitro. Pol I was first incubated with the template in the presence of purified factors, UAF, CF, TBP, and Rrn3, followed by the addition of ATP, UTP, GTP, and [α-32P]GTP but without CTP. Then heparin was added to prevent reinitiation of transcription. Finally, CTP was added to allow the paused Pol I (presumed to be at +56, which precedes the first base pair encoding C) to continue elongation. Accumulation of the full-length runoff product (763 nt) was then monitored over time (). From these data we calculated that the WT elongation rate at room temperature (~20°C) was ~30 nt/s, whereas the mutant enzyme was ~10-fold slower. These data clearly demonstrate that the rpa135(D784G) mutation impaired transcription elongation compared to WT. (For technical reasons, we were not able to clearly observe the stalled +56 complex on our gel; however, because equal amounts of WT and mutant Pol I yield approximately equal amounts of product, the mutant Pol I does not arrest or terminate prematurely.)
Defects in Ribosome Assembly Caused by the rpa135(D784G) Mutation
To measure effects of the rpa135
mutation in vivo, the mutation was introduced into the chromosomal RPA135
gene in our standard strain (NOY388; “WT”), yielding the mutant strain NOY2172. Both the mutant and the WT strains were grown exponentially in synthetic glucose complete (SD) medium at 30°C. The growth rate of the mutant was about 55% of the growth rate of the WT strain. In sucrose gradient sedimentation analysis, the mutant extract showed a significant decrease in the amount of free 60S relative to free 40S subunit and an appearance of “halfmers” which represent 80S monosomes or polysomes containing an additional 40S subunit (Helser et al., 1981
; ). This pattern suggested a preferential reduction in 60S relative to 40S subunits (Rotenberg et al., 1988
). To confirm that this anomalous ribosome profile was not a consequence of a general decrease in Pol I transcription rate, we examined the ribosome profile of a strain in which Pol I transcription initiation was specifically impaired by mutation of the transcription initiation factor Rrn3 (rrn3[S213P]
; Claypool et al., 2004
). When grown at the semipermissive temperature (30°C), this strain grew slightly slower than the rpa135
strain; however, the ribosome profile appeared normal (). Thus, only when Pol I transcription elongation is impaired do we observe defects in ribosome assembly.
rRNA Accumulation and Ribosome Assembly Are Impaired in the rpa135(D784G) Mutant Compared to WT
To quantify effects of the rpa135 mutation on individual RNA species, we grew cells in SD −Ura medium containing [14C]uracil for many generations and measured total RNA (cold-TCA-precipitable 14C-labeled RNA) as well as individual species excised after gel electrophoresis. The results are summarized in . Calculation showed that even though cell growth rate in the mutant was significantly reduced due to a strong (~4.0-fold) decrease in the rate of production of mature rRNA species by Pol I, no decrease in the amount of tRNA (per cell mass) was observed. In contrast to tRNA, the amount of Pol III-derived 5S rRNA in the rpa135 mutant was significantly lower than in WT cells (per cell mass), similar to the levels of Pol I-derived rRNA species. It is likely that 5S rRNA is synthesized normally by Pol III in the rpa135 mutant, but because fewer functional ribosomes are assembled due to impaired Pol I elongation, excess 5S rRNA is degraded.
We also observed a small but reproducible decrease (~20%) in the amount of 25S rRNA relative to 18S rRNA in the mutant strain compared to WT (). This observation is consistent with the ribosome profiles in . Finally, there was a reduction in the amount of 35S pre-rRNA in the mutant cells compared to WT, consistent with experiments described below. All of these data support the conclusion that impairment of Pol I elongation affects ribosome assembly.
Defects in rRNA Processing Caused by the rpa135(D784G) Mutation
To examine defects in specific rRNA processing steps, we analyzed the abundance of various pre-rRNA species in the WT and rpa135
cells by northern blot and primer extension. Because it has been shown previously that polyadenylated forms of normal and aberrant rRNA processing intermediates accumulate when the function of the nuclear exosome is disrupted (Kuai et al., 2004
; Fang et al., 2004
; Houseley et al., 2006
), we included two additional strains in these studies. One strain contained a deletion of the gene for the nuclear-specific exosome subunit Rrp6 in our WT strain background (NOY2175). The other strain was a double mutant: rrp6 rpa135
We observed abnormal processing of 35S pre-rRNA leading to mature 18S, 5.8S, and 25S rRNAs in the rpa135 mutants. First, there was a large reduction in the amount of the 35S pre-rRNA in the two strains carrying the rpa135 mutation compared to control strains (, compare even-numbered lanes with odd-numbered lanes). This result is consistent with the results obtained by direct analysis of cellular RNAs fully labeled with [14C]uracil mentioned above (see ).
The rpa135(D784G) Mutation Causes Defects in rRNA Processing and Accumulation of Aberrant Pre-rRNA Species
Processing in the 5′ ETS region of 35S pre-rRNA is abnormal in the rpa135
strains. Normally, cleavage at A0
releases an ~0.6 kb fragment that is degraded by the TRAMP [Tr
olyadenylation complex]-exosome complex (de la Cruz et al., 1998
; Allmang et al., 2000
; Zanchin and Goldfarb, 1999
; for TRAMP, see Kadaba et al. 
, LaCava et al. 
, Vanacova et al. 
, Wyers et al. 
, and Houseley et al. 
). We observed accumulation of this 5′-A0
fragment in the rrp6
strain, consistent with this model (, lane 3). However, this fragment is diminished in the rrp6 rpa135
double mutant (, lane 4) demonstrating that impaired Pol I elongation resulted in either a large decrease in normal cleavage at A0
or additional abnormal cleavage events. These data are supported by primer extension analyses which showed that rpa135
cells contain more RNAs with intact 5′ ends (, lanes 1–4) but have less properly processed A0
ends (, lanes 5–8) when quantified per total RNA. Thus, processing at A0
is impaired in the rpa135
Processing of the 18S rRNA is further impaired in the rpa135 mutant cells. With both probes “a” (for 5′-A0) and “b” (for A0–A1), we observed increased intensities of a smear from ~0.8 kb to ~2 kb in the strains carrying the rpa135 mutation. Within this smear, several bands were observed and an ~1 kb band was especially strong in the rrp6 rpa135 strain (, lanes 4 and 8; indicated as “5′-S1”). These fragments result from endonucleolytic cleavage(s) within the 18S region of pre-rRNA. They contain most (or all) of the 5′ ETS as well as proximal 18S rRNA (i.e., they were not cleaved at either A0 or A1). The accumulation of these aberrant cleavage products explains why the production of mature 18S rRNA (per total RNA) () and its normal precursors 20S rRNA (, lanes 9–12) and 35S rRNA ( and ) is reduced in the rpa135 mutants even though we detect higher amounts of the 5′ end (i.e., +1) of pre-rRNA in these mutants than in WT by primer extension (, lanes 1–4). For convenience in discussion, we call the major aberrant cleavage site (or sites, see below) responsible for ~1 kb RNA fragment “S1” (small subunit rRNA cleavage site 1; ). We note that a fragment corresponding to 5′-S1 can be detected weakly in WT cells. Thus, it appears that this aberrant cleavage occurs at a very low frequency normally, the rpa135 mutation causes a large increase in this frequency, and the TRAMP-exosome complex plays a role in eliminating this aberrant product.
Processing events leading to formation of 5.8S rRNA were also found to be aberrant in the rpa135
strains. With probe “d” (for A2
), an ~0.4 kb band was clearly observed in the rpa135 rrp6
strain (, lane 16, “A2
”), but only weakly in the rpa135
strains and not in the WT strain (, lanes 13–15). The same signal was observed with a probe for the A3
region (data not shown), but not with probe “e” (for C2
) (, lane 20) in the rpa135 rrp6
strain. This band likely corresponds to the A2
fragment, which was previously observed in mutants defective in processing factor Ssf1 (Fatica et al., 2002
) or Npa1 (Dez et al., 2004
), in cells depleted of exosome components (Allmang et al., 2000
) or those treated with 5-fluorouracil (Fang et al., 2004
). This fragment is apparently produced by premature cleavage at C2
prior to the cleavage at A3
(Fatica et al., 2002
Another feature we observed for the rpa135
strain is an increase of the ~3 kb RNA relative to WT detected by probes a, b, c, and d (but not by e), which was also increased by the rrp6
mutation (; indicated as 23S). This RNA must include most of the 5′ ETS through A3 region and may correspond to the 23S pre-rRNA that is known to be produced by the cleavage at A3 as an alternative pathway when cleavages at A0
, and A2
are inefficient (Venema and Tollervey, 1999
Finally, we observed degradation of precursors for 25S rRNA. With the A2–A3 and C2–C1 probes (d and e), all of which detected the presence of 27S pre-rRNA (~3.9 kb), we observed increased intensities of several bands and smears ranging from ~0.7 kb to ~2 kb in both the rpa135 and rpa135 rrp6 strains relative to controls (, lanes 13–20). These fragments likely contain the A2 site as their 5′ end, with various sites within 25S rRNA as their 3′ end, since we detect more RNA with an A2 5′ end in the rpa135 mutant cells by primer extension (, lanes 9–12) but equal or less 27S pre-rRNA in the rpa135 mutants than in control strains (, lanes 13–20). These results indicate that the rpa135 mutation impairs the normal processing pathways for production of the 25S rRNA via 27S pre-rRNA by causing cleavages (and degradation) within the region corresponding to the mature 25S rRNA.
Aberrant RNA Fragments Are Polyadenylated
Preliminary experiments suggested that the aberrant RNA species generated in the rpa135
mutant were polyadenylated, indicating a potential role for the TRAMP complex, which is involved in rRNA quality control (LaCava et al., 2005
; Houseley et al., 2006
). We confirmed this prediction for the aberrant 5′-S1
RNA. As shown in , poly(A) tailed RNAs were detected first by reverse transcription using an oligo(dT)-adaptor primer, followed by PCR amplification of the products using a primer with the 5′ end at +674 combined with the adaptor portion of the primer used for the reverse transcription (, lanes 1–4). From the size of the band (~300 bp), the major cleavage site (S1
) was calculated to be ~270 nt from the 5′ end of the mature 18S rRNA. PCR products obtained in these experiments were cloned and sequenced to identify cleavage/polyadenylation sites (). Although there is an indication of a “hot spot” around position 262, other cleavage/polyadenylation sites were also identified. We conclude that cleavage or cleavages followed by polyadenylation take place within the 18S region during processing of precursor rRNA; these RNAs are degraded by the nuclear exosome, and the frequency of such cleavage/degradation of precursor rRNA is increased in the rpa135
mutant strains. Similar cleavage/polyadenylation of precursor rRNA followed by degradation also takes place within the 25S region, but we have not characterized these events in detail.
The 5′-S1 Fragment Is Polyadenylated
The rpa135 Mutant Polymerase Is as Processive as WT
To confirm that the aberrant RNA transcripts observed (–) resulted from abnormal cleavage/processing of the rRNA rather than premature termination of transcription by the mutant polymerase, we measured the processivity of Pol I using two methods: (a) biochemical analysis of Pol I processivity using a chromatin immunoprecipitation (ChIP)-based assay and (b) direct visualization of transcribing Pol I molecules by EM analysis of Miller chromatin spreads.
Our ChIP assay for processivity compares Pol I association with the 35S rRNA gene near the 5′ end to that near the 3′ end (Mason and Struhl, 2005
; Schneider et al., 2006
). Pol I (and crosslinked DNA) was immunoprecipitated with a polyclonal antibody against A190 followed by extensive washing and reversal of the crosslinks. The ratio of ChIP signal (IP divided by input) at the 3′ end relative to the 5′ end can be used to estimate the “relative processivity” of Pol I in a given strain (). When quantified and normalized to WT, it is clear that there is no decrease in the relative processivity of Pol I in the rpa135
mutant cells (, right panel). These data indicate that defects in rRNA processing/ribosome assembly cannot be explained by defects in processivity, in vivo.
Processivity of Pol I Is Not Affected by rpa135 Mutation
EM Miller chromatin spread analysis of the WT and rpa135 mutant cells further confirmed that there are no processivity defects in the mutant strain. If the rpa135 mutant polymerase were more prone to premature termination, then EM analysis would detect differences in the polymerase density near the 5′ end of the gene versus the 3′ end compared to WT. As seen in both the representative EM pictures () and the quantification of Pol I occupancy within the four quarters of the gene for a larger sampling of genes (), there is no evidence for increased premature termination or release of Pol I in the mutant compared to WT. These data support the interpretation that elongation defects of the mutant polymerase cause defects in rRNA processing and ribosome assembly.
In addition to confirming the absence of polymerase processivity defects, EM analysis revealed several other features of rRNA transcription in the mutant. First, by quantification of the polymerase density of a large number of genes, we observed an ~30% decrease in the average polymerase density per gene in the rpa135 mutant compared to WT (33 Pols/gene compared to 49 Pols/gene in WT; ). Thus, the rpa135 mutation decreases the rate of initiation ~30% more than the overall rate of elongation in vivo. However, because defects in initiation in the rrn3 mutant do not cause defects in rRNA processing/assembly (), the defect in initiation caused by the rpa135(D784G) mutation cannot be responsible for the observed defects in rRNA processing/assembly observed in this mutant.
The micrographs also showed that transcripts attached to Pol I seen in the rpa135 mutant are often short or missing relative to those at similar positions within a gene in the WT strain (examples shown by arrows in ), suggesting that the anomalous pre-rRNA cleavages/degradation observed for the mutant ( and ) take place cotranscriptionally. Such cotranscriptional defects are consistent with the conclusion that efficient rRNA processing/ribosome assembly requires proper transcription elongation. This cotranscriptional rRNA degradation is being investigated further.