S1 nuclease protection analysis of RNA 3′ end formation on the chromosomal ribosomal genes.
A common laboratory strain of yeast (W3031a) was grown to mid-log phase in rich medium, and total RNA was extracted. Aliquots of RNA were then hybridized with a single-stranded, end-labeled DNA probe which has a labeled 3′ terminus at −72 relative to the 3′ end of 25S rRNA and extends to +179, where its sequence diverges from the sequence of the rDNA in strain W3031a (probe 1; termini illustrated in Fig. ). The hybrids were digested with S1 nuclease, and protected probe fragments were displayed on a denaturing urea-acrylamide gel as shown in Fig. A, lane 1.
FIG. 2 (A) S1 nuclease analysis of RNA 3′ ends formed on the chromosomal rDNA of strain W3031a. In lane 1, probe 1 was hybridized to total cellular RNA and digested with S1 nuclease, and the protected fragments were displayed on a denaturing acrylamide (more ...)
Probe protection due to the mature 3′ end of 25S rRNA is the predominant signal in lane 1, and we designate the leading edge of this cluster of bands as position −1 in the numbering scheme shown in Fig. . The next most prominent bands are in a cluster located from about +72 to +93 beyond the 3′ end of 25S. A higher-resolution gel of the +72 to +93 cluster (Fig. B) shows that the longest band corresponds to position +93. This is the same location as identified previously as the site of PolI termination at the Reb1p terminator by in vitro studies (6
). The shorter bands in the +72 to +93 cluster probably represent 3′ processing products of primary terminated RNA. 3′ processing of terminated transcripts has been observed in mammals (10
) as well as in amphibians (12
), and we have observed 3′ processing of terminated and released transcripts in yeast whole-cell extracts (16a
). As shown below (Fig. ), in vivo mutagenesis of the Reb1p terminator is consistent with the +93 band representing paused transcripts whereas the shorter transcripts may be released and 3′ processed transcripts. When quantitating termination at the +93 site, we routinely sum all of the bands in the +72 to +93 cluster and compare their intensities to the intensity of the cluster at −1.
In Fig. A, lane 1, the only significant band beyond the +93 cluster is a faint band located at +179. This is the point where the sequence of probe 1 diverges from the rDNA sequence of strain W3031a. The practical result of this divergence is that all transcripts which read through the Reb1p terminator are truncated at this point and collected into a single band. In addition, if any rDNA contamination survived the RNA purification, probe protection due to this contaminant could contribute to the +179 band. PhosphorImager quantitation of Fig. A and similar experiments shows that the intensity of the 3′ 25S signal is 200- to 300-fold greater than the intensity of the +93 cluster of bands. The intensity of the +179 readthrough band ranges from 1 to 10% of the +93 signal in different experiments. We conclude from this experiment that RNA termini corresponding to the Reb1p terminator are readily detectable in wild-type yeast cells. Furthermore, this analysis detects 10% or less readthrough past the +93 site, consistent with it being the primary terminator of PolI transcription.
Quantitation of PolI termination by RT-PCR.
S1 nuclease analysis is useful for precisely locating RNA termini. However, accurate quantitation of PolI termination efficiency by this method is difficult due to the low amount of RNA extending to +93 in the presence of a much larger amount of 25S rRNA. We initially thought to circumvent the problem of excess 25S rRNA by use of a shorter DNA probe whose labeled 3′ end would be located beyond the 25S sequence. After several failures with such a probe, we realized that a large stable hairpin is present in the RNA in this region (this is the hairpin recognized by RNase III during the processing that forms mature 25S rRNA [4
]). The labeled terminus of any practical short probe must compete for hybridization against the formation of a more stable RNA-RNA hybrid.
Ultimately we decided to use RT-PCR to measure PolI termination efficiency. As illustrated in Fig. A, we first synthesized four short primers, P1 to P4. An excess of each primer was hybridized to a separate, uniform aliquot of total yeast RNA and was then extended with reverse transcriptase to make cDNA. The relative abundance of cDNA extending from each primer position was then measured by PCR amplification using a radioactive common 5′ primer (P5) paired with the same 3′ primer originally used to make each cDNA. Parallel amplification reactions were run with each primer pair, and aliquots were removed every five cycles to measure the progress of amplification. As shown in Fig. B, the product of the P1-P5 primer pair, corresponding to the 3′ end of 25S rRNA, amplifies rapidly and uses up the radioactive P5 primer by 15 cycles. Amplification by the P2-P5 primer pair, corresponding to cDNA extending to +93, proceeds more slowly, consistent with +93 transcripts being several orders of magnitude less abundant than 25S rRNA. The P3-P5 primer pair (detecting cDNA from transcripts extending beyond +93) amplifies even more slowly, consistent with most transcripts stopping at +93. Finally, primer pair P4-P5 barely makes any product, even at 30 cycles. The small amount of product amplified by P4-P5 is about the same as is amplified from a mock cDNA reaction in which either the reverse transcriptase or the P4 primer was omitted. Thus, this small amount of product is probably due to residual DNA in the RNA preparation.
The RT-PCR data in Fig. B are qualitatively in agreement with the S1 nuclease analysis shown in Fig. A. They confirm that transcripts extending to +93 are less abundant than 25S rRNA and that most transcripts do not extend beyond the +93 site. However, the more sensitive RT-PCR method verifies that there is detectable readthrough past the +93 site, and furthermore, this readthrough does not extend beyond +250. Beyond +250, the PCR method detects only a trace of DNA contamination that escaped DNase treatment during the initial RNA preparation.
To determine absolute transcript abundances, we used the same cDNAs and primer pairs to perform competitive PCR. As shown in Fig. A, we constructed a DNA internal standard which has a 75-bp insert of plasmid DNA situated so that it will be amplified by all primer pairs. Known amounts of this internal standard DNA were then mixed with each of the cDNAs, and both cDNA and internal standard were amplified with the appropriate primer pair in the same reaction. When the amplified signal obtained from both the cDNA and the internal standard were the same, we assumed that the amount of internal standard added matched the amount of cDNA (Fig. C). In cases where the internal standard did not exactly match the cDNA, we interpolated between the two nearest internal standard amounts to estimate cDNA concentration. If we assumed that most of the stable RNA in a yeast cell is 17S and 25S rRNA and that reverse transcriptase synthesized a single cDNA molecule for each molecule of 25S rRNA, then we added 0.075 pmol of 25S cDNA to the P1-P5 amplication. As shown in Fig. C, we estimated by competitive PCR that about 0.15 pmol of 25S rRNA was in the reaction, a difference of only twofold from the first estimate.
The results of competitive PCR quantitation are shown in Table . We estimated on the basis of S1 nuclease protection (Fig. A) that 25S rRNA is about 300-fold more abundant than transcripts extending to +93, while the estimate by PCR is 3,000-fold difference. It is likely that the lower estimate by S1 analysis is due to the fact that the experiment was not done under conditions of probe excess and the PCR estimate is the correct estimate. Quantitative PCR agrees with S1 analysis that about 90% of transcripts stop at +93 with about 10% readthrough beyond this point. No transcripts extend beyond +250.
TABLE 1 Quantitation of transcripts in the PolI termination region byRT-PCR PolI termination in an RNase III temperature-sensitive rnt1-47 strain.
The low abundance of transcripts extending beyond the 3′ end of 25S rRNA makes it difficult to study their termini. Recently it has been shown that the stability of these transcripts can be greatly increased by inactivation of the RNT1
gene product, an RNase III which is involved in the processing steps that form the 3′ end of mature 25S rRNA (4
). Figure A shows S1 nuclease protection analysis of RNA from strain HI227 (rnt1-47
), a strain that carries a temperature-sensitive mutation in RNT1
(a generous gift from M. Ares). RNA was isolated from HI227 cells grown at the permissive temperature (24°C) and at intervals after shifted to the nonpermissive temperature (37°C). S1 protection was done with the same probe (probe 1) as used previously for Fig. A.
FIG. 4 S1 nuclease analysis of RNA 3′ ends formed on the chromosomal rDNA of strain HI227 (rnt1-47). (A) Total cellular RNA was extracted from HI227 cells grown either at 24°C (lane 1) or at intervals after a shift to 37°C (lanes 2 to (more ...)
S1 analysis of RNA from HI227 grown at the permissive temperature (Fig. A, lane 1) looks similar to that seen with RNA from wild-type W3031a analyzed at a similar probe-to-RNA ratio (Fig. A, lane 1). In RNA from HI227, a cluster of bands is seen in the +72 to +93 region and the apparent readthrough signal at +179 is about 6% of the +72 to +93 cluster intensity. However, shifting HI227 to 37°C has a dramatic effect on 3′ end formation (Fig. A, lanes 2 to 4) which is graphically summarized in Fig. B. The intensity of the 3′ 25S signal decreases, the intensity of the +72 to +93 cluster increases nearly to that of the 3′ 25S signal, and readthrough to the +179 divergence point also increases dramatically. Increased stability of the longer transcripts now makes it easy to detect events at the Reb1p terminator and beyond.
A fail-safe terminator beyond the Reb1p terminator.
In contrast to the situation in a wild-type cell, inactivation of Rnt1p results in considerable readthrough at the Reb1p terminator. We used probe 2 (Fig. ) to determine where these readthrough transcripts terminate. Probe 2 was derived by PCR from the genomic rDNA of strain W3031a and matches the rDNA sequences of both W3031a and HI227 to a HincII site at position +415 beyond the 3′ end of 25S rRNA. Probe 2 has additional vector sequence beyond the divergence point to allow full-length transcripts to be distinguished from undigested probe. Figure C shows an S1 protection assay in which probe 2 was hybridized with RNA from HI227 cells grown at 37° for 4.5 h. The S1 protection pattern shows bands corresponding to 3′ 25S and the +72 to +93 cluster similar to bands seen with probe 1 in the same RNA sample (Fig. A, lane 3). In Fig. C, however, readthrough past the Reb1p terminator appears as a cluster of bands of which the longest maps to a position about +250 bp beyond the 3′ end of 25S. Quantitation of Fig. C shows that no transcripts are detectable beyond the +250 site to the point where probe 2 diverges from rDNA sequence at +415. Therefore, +250 appears act as a completely efficient fail-safe terminator for transcripts that escape the Reb1p terminator.
In their original analysis of RNA synthesized after heat shock of a rnt1
temperature-sensitive strain, Elela et al. observed a disappearance of signal from 3′ 25S rRNA and saw no evidence of termini at +93 (4
). Instead, they saw only the appearance of a cluster of bands mapping approximately to +250. We have been able to duplicate these results by using a small amount of very hot probe and doing the S1 nuclease analysis under conditions of large RNA excess (data not shown). These are conditions predicted by Lopata et al. (17
) to favor formation of triple and higher-order hybrids in which the longest transcripts in the mixture suppress detection of shorter transcripts. When the amount of probe is increased the amount of RNA is decreased, termini corresponding to 3′ 25S and +93 are readily detected in RNA from rnt1
cells (Fig. ).
Termination on a ribosomal minigene, 105LT.
We have previously assayed a series of mutants of the Reb1p terminator for their effects on PolI termination in vitro (15
). To test these same mutations in the living cell, we constructed a ribosomal minigene, 105LT (Fig. A). 105LT has an entire intergenic spacer region, including a PolI promoter and enhancer, upstream of a 314-bp minigene. The minigene contains a unique Xho
I site at +36 to allow use of primers to specifically monitor transcription initiation from the minigene promoter. The Xho
I site also allows radioactive end labeling of a single-stranded DNA probe (probe 3) to measure RNA 3′ end formation on 105LT by S1 nuclease protection. The Reb1p termination site is 285 bp downstream of initiation, and the terminator consists of a 53-bp fragment of rDNA that was previously found to direct wild-type levels of termination in vitro (16
). The 53-bp terminator fragment contains 23 bp of T-rich sequence upstream of termination plus 30 bp downstream (including the Reb1p binding site). Probe 3 contains 38 nt of additional length beyond the point where it diverges from the sequence of 105LT to allow readthrough transcripts to be distinguished from undigested probe.
105LT was transformed into W3031a on a 2μm yeast vector. Figure B, lanes 2 and 3, shows the patterns of S1 protected bands obtained with RNA from two independent transformants. In each lane, there is a band at +247 that we interpret as the primary termination product. Extending down from +247 is a cluster of bands which presumably represent 3′ processing products of the primary terminated transcript. As an internal size standard, we have cut the plasmid used to make S1 probes with BfrI and transcribed the truncated DNA with T7 RNA polymerase. This is expected to yield an RNA that is 3 nt longer than the primary termination product that we observe in purified in vitro termination systems. The band, labeled +247, migrates detectably faster (lanes 2, 3, 5, and 6) than the band protected by the BfrI transcript (lane 8) and thus, within the resolution of this gel, maps to the location previously mapped in vitro as the primary site of Reb1p- directed termination.
Termination on 105LT is leaky. PhosphorImager analysis, summarized at the bottom of Fig. B, shows that about 33% of total transcripts read past the Reb1p terminator and appear as a band of 284 nt (lanes 2 and 3).
Effect of mutating the Reb1p terminator on PolI termination in vivo.
Previous in vitro work has identified three informative mutants of the Reb1p terminator. The sequence of these mutations is shown in Fig. C. Mutant 1 changes two G residues in the Reb1p binding site to T’s. This alteration eliminates Reb1p binding as assayed by gel shift analysis and concurrently eliminates PolI termination in vitro (15
). When the same mutations are introduced into 105LT, they also abolish all termination in vivo with a corresponding increase in readthrough (Fig. B, lane 4).
Mutant 2 changes three A residues in the Reb1p binding site to C’s. When assayed in vitro these changes increase the binding of Reb1p about 10-fold and also increase pausing and overall termination efficiency (15
). Introduction of these changes into 105LT has a similar effect in vivo since it decreases readthrough from about 33% to about 13% of total transcripts (lane 5).
Mutant 3 changes three T residues to G’s within the upstream T-rich element of the Reb1p terminator. In vitro work indicates that this change inhibits transcript release while having little effect on pausing caused by Reb1p binding (16
). It is a reasonable interpretation that the primary termination product at +247 represents transcripts that have paused but not released from the template. Conversely, protected bands shorter than +247 are likely the result of 3′ processing of released transcripts. At a wild-type terminator, the ratio of primary to processed products is 0.3 to 0.4 (Fig. B, lanes 2 and 3). When the three G substitutions are introduced into the upstream T-rich element, however, this ratio changes dramatically to 1.7 (lane 6). This change in ratio is consistent with transcript release being impaired by mutant 3 in vivo, similar to its effect in vitro.
Analysis of these three mutant terminators supports the conclusion that termination directed by Reb1p proceeds in vivo by essentially the same mechanism as previously defined by test tube studies (21
). Termination requires the Reb1p binding site, increasing the affinity of this site for Reb1p also increases termination efficiency, and introducing G residues into the T-rich upstream element impairs transcript release. Primer extension analysis (data not shown) reveals that none of the terminator mutants has any effect on transcription initiation by PolI.