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Proc Natl Acad Sci U S A. 2006 February 28; 103(9): 3262–3267. Published online 2006 February 16. doi: 10.1073/pnas.0507783103. | PMCID: PMC1413877 |
Copyright © 2006 by The National Academy of Sciences of the USA Genetics Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae Carrie Anne Davis and Manuel Ares, Jr.* Center for Molecular Biology of RNA, Department of Molecular, Cell, and Developmental Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064 Received September 8, 2005. Freely available online through the PNAS open access option. Mutations in RRP6 result in the accumulation of
aberrant polyadenylated transcripts from small nucleolar RNA genes. We
exploited this observation to search for novel noncoding RNA genes in
the yeast genome. When RNA from rrp6Δ yeast
is compared with wild-type on whole-genome microarrays, numerous
intergenic loci exhibit an increased mutant/wild type signal ratio.
Among these loci, we found one encoding a new C/D box small nucleolar
RNA, as well as a surprising number that gave rise to heterogeneous
Trf4p-polyadenylated RNAs with lengths of ≈250–500 nt.
This class of RNAs is not easily detected in wild-type cells and
appears associated with promoters. Fine mapping of several such
transcripts shows they originate near known promoter elements but do
not usually extend far enough to act as mRNAs, and may regulate the
transcription of downstream mRNAs. Rather than being uninformative
transcriptional “noise,” we hypothesize that these
transcripts reflect important features of RNA polymerase activity at
the promoter. This activity is normally undetectable in wild-type
cells because the transcripts are somehow distinguished from true
mRNAs and are degraded in an Rrp6p-dependent fashion in the
nucleus. Keywords: compound promoters, cryptic RNAs, genome annotation, RNA decay The ability to sequence complete genomes has demanded comprehensive
approaches to gene discovery and genome annotation, often using
computational predictions. Eventually, experimental data must be used
to assign gene function to the genome. Because transcription is the
primary biosynthetic event required for gene function, identifying
transcribed regions is the foundation of genome annotation. Numerous methods allow the genome-scale annotation of transcribed
regions. For example, cDNA cloning strategies or serial analysis of
gene expression (SAGE) capture evidence of transcription not dependent
on prior information, such as ORF prediction ( 1). However, transcripts present in low abundance may
be missed unless cloning biases are controlled and exhaustive numbers
of clones are studied. More recently, “whole genome” or “genomic
tiling” microarrays have been used to identify transcribed
regions ( 2, 3). Such arrays have the advantage of
not relying on the current genome annotation and are not limited by
“clone coverage” problems of EST or serial analysis of
gene expression approaches. Because of this, they have been used to
map RNAs from the human ( 2, 4) and other genomes ( 5– 7). One
limitation to this approach is its sensitivity, which depends on how
well an expressed RNA can be captured by the microarray. This property
is likely governed by combined effects of the abundance of the
transcript in the sample, its labeling efficiency, and the ability of
probes on the array to capture the labeled target. Based on our observations of global changes in RNA levels for
introns in dbr1Δ yeast and for small nucleolar
RNA (snoRNA)-containing introns in yeast using the
rrp6Δ mutation ( 8, 9), we
reasoned that locating genes or gene elements whose processing or
decay requires specific factors (e.g., the debranching enzyme for
introns, or Rrp6p for snoRNAs) could be accelerated by combining
whole-genome microarrays with mutants in RNA processing pathways.
Array elements representing genomic regions that produce transcripts
whose abundance or labeling efficiency depend on proper RNA processing
would be expected to capture different amounts of target in mutant vs.
wild type comparisons, thus revealing their location. To test this
idea, we searched for new small RNAs using strains lacking Rrp6p, a
component of the nuclear exosome responsible for 3’ end
processing of small stable transcripts such as small nuclear RNAs
(snRNAs), snoRNAs, and rRNAs ( 10). Mutations in RRP6 lead to the
accumulation of snRNAs, snoRNAs, and rRNAs with 3’ extensions
and poly(A) tails ( 11– 13), thus increasing their labeling efficiency in the
mutant sample. In this article, we compare RNA from wild type cells with RNA from
a strain lacking RRP6 using microarrays containing
the complete genome. Recently, an approach using arrays that carried
probes only for previously discovered RNA transcripts found that many
intergenic RNAs represented by serial analysis of gene expression
transcripts are increased in the rrp6Δ mutant
( 13). Because we used arrays
covering the entire genome, we discovered numerous regions not
previously known to be transcribed. In one such region, we uncovered a
new C/D box snoRNA, as we had hoped. The other class of RNA was
unexpected and consists of transcripts that appear to originate from
promoter regions of standard protein-encoding genes. In wild-type
cells, these transcripts are decayed through a Trf4p-Rrp6p pathway
( 13– 16). We propose that these
transcripts are produced at mRNA promoters across the genome as a
consequence of RNA polymerase II activity that normally does not lead
to functional mRNA. Expression of RNAs from Many Genomic Loci Increases upon Loss
of RRP6. Our initial goal was to use the observations of Van Hoof
et al. that noncoding RNAs
accumulate as polyadenylated species ( 11) and a whole-genome microarray ( 17) to identify new stable RNAs
across the entire yeast genome ( A). Because reverse transcriptions were
primed with a mixture of oligo(dT) and random hexamers, array signals
should increase for transcripts whose poly(A) content might change
upon loss of Rrp6p, even if the overall level of RNA does not, due to
more efficient Cy-dye labeling of poly(A)+ RNAs in the presence
of oligo(dT). After normalization, we find that 97% (12,754) of the total 13,137 array features have
log 2 ratios within two standard deviations
(SD = 0.5) of the mean (mean = +0.03) ( B, white bars).
However, 2.6% (350) of the loci exhibit
log 2 ratios more than 2 SDs above the mean
in the rrp6Δ mutant (Table 1, which is
published as supporting information on the PNAS web site). This group
is dominated (51/350) by known snoRNAs ( B, black bars; 20 known snoRNAs
are below this cutoff) or regions 3’ of known snoRNAs (28/350;
Table 2, which is published as supporting information on the PNAS web
site). Spliceosomal snRNAs U4 and U6 were also among those detected.
Although polyadenylation of rRNAs occurs in the
rrp6Δ mutant ( 12), we observed no increase in signal for rRNA
features, possibly because rRNA is saturated. These results are
consistent with reports indicating that snoRNAs and snRNAs accumulate
as polyadenylated products in the rrp6Δ mutant
( 10, 11) and demonstrate that small stable RNA loci in the
genome can be easily distinguished by using whole-genome microarrays
and an rrp6Δ mutant strain. To begin
identifying new small RNAs, we studied several of the ≈160
unannotated intergenic loci in this group ( B, gray bars). A New C/D Box snoRNA Is the Guide for 18S rRNA Methylation at
Position A436. Intergenic region iYKL006c-a gave a 6-fold increase in signal in
the rrp6Δ mutant. Northern blots probed with
this region uncovered a small stable RNA ≈106 nt in length
( A, lane
1). This RNA appears slightly longer in the
rrp6Δ strain (lane 2), consistent with the
proposed role for Rrp6p in trimming the last three nucleotides ( 10, 11). In addition, a slower migrating heterogeneous
RNA accumulates ( A, lane 2). This RNA binds oligo(dT)
indicating that it is polyadenylated (data not shown), as are other
snoRNAs in strains lacking RRP6 (see below) ( 11). We mapped this RNA, called
snR87, to the Crick strand of chromosome XI between positions 430850
and 430670, a highly conserved region in related yeasts (Fig. 7, which
is published as supporting information on the PNAS web site). Using an
rnt1Δ strain, we find that the 5’ end
of this snoRNA is generated in part by Rnt1p processing, as for other
snoRNAs (Fig. 7) ( 18, 19). The sequence of snR87 contains C/D box snoRNA motifs and
complementarity to 18S rRNA, characteristics of methylation guide RNAs
for rRNA ( 20) ( C). The rRNA
complementary region suggested that snR87 might guide methylation of
A436, an rRNA methylation site without a known C/D box snoRNA. To test
this, we compared methylation at A436 in wild type with a strain
deleted for snR87 ( B). In wild type, reverse transcriptase stops
occur at positions just downstream of the known methylation sites at
A436 and A420 (lanes 4–6). In contrast, no stop consistent with
methylation is seen at A436 in the snR87Δ
strain, although the stop at A420 is readily apparent (lanes
1–3). This demonstrates the requirement for snR87 in 2’-O-methylation of 18S rRNA position A436 and validates this genomic
approach for finding noncoding RNAs by testing the prediction of van
Hoof ( 11) on a genomic
scale. Many Genomic Regions Encode Heterogeneous RNAs That Appear
Unstable in the Presence of Rrp6p and Trf4p. Probing of other regions uncovers another class of noncoding RNA
distinct from snoRNAs, snRNAs, and rRNAs ( A, lane 2). Accumulation of
these RNAs, which range in size from 250 to 800 nt, is caused by loss
of RRP6 because no RNA was seen in the wild-type
strain. Furthermore, each of these RNAs is enriched in oligo(dT)
selected RNA (lane 3), indicating that they are polyadenylated in the
rrp6Δ mutant. Similar findings ( 13) were recently reported for
regions of the yeast genome previously shown to be transcribed by
using serial analysis of gene expression ( 1). Recently, a decay complex now referred to as TRAMP was described
( 13– 16). Trf4p is a component of this
complex that acts by adding 10–40 adenines onto the 3’
end of RNAs before their decay by the exosome ( 13– 16). To test whether loss of Trf4p could cause
accumulation of the RNAs we identified, we blotted RNA from a
trf4Δ strain and probed with several genomic
regions. RNA from each of these regions is stable in the
trf4Δ mutant ( B, lane 2) and is not enriched
in poly(A)+ fractions (lane 4), in contrast to the
rrp6Δ mutant. These results are consistent
with a model in which Trf4p marks this class of RNAs for nuclear decay
through addition of a polyA tail followed by Rrp6p-mediated
degradation (see ref. 13). The SER3 Promoter-Associated SRG1 RNA Accumulates in the
rrp6Δ Mutant. SRG1 RNA is a noncoding promoter-associated RNA whose expression
regulates the adjacent SER3 gene ( 21). We asked whether the
SRG1 RNA accumulates in the
rrp6Δ mutant. Although the
SER3 promoter region encoding SRG1
RNA did not exhibit a change in signal log 2
ratio, the SER3 ORF 3’ of
SRG1 increased 4-fold in the
rrp6Δ mutant, comparable to the 3’
regions of the snoRNA genes (Table 2). Transcription of
SRG1 spans the SER3 promoter and
extends into the 5’ end of the SER3 coding
region ( 21). We saw significant
accumulation of SRG1 RNA in the
rrp6Δ strain compared with wild type on a
Northern blot ( A). Although SRG1 RNA is
reported to be polyadenylated ( 21), we did not observe efficient enrichment in the
poly(A)+ fraction from wild type using our method ( A), although
there is a small increase in the amount of polyadenylated
SRG1 the rrp6Δ mutant. In
contrast, snR128 extended transcripts are well selected on oligo(dT)
( A,
compare lane 2 with lane 5 for each RNA). For both RNAs the loss of
Trf4p leads to more modest accumulation of RNA that is not enriched in
the poly(A)+ fraction ( A). We conclude that SRG1
RNA decay occurs via the exosome. Mutations in components of the chromatin remodeling machinery lead
to SER3 induction and a partial loss of
SRG1 RNA accumulation ( 22), as well as an increase in aberrant transcripts
that appear to originate at promoter-like sequences within coding
regions ( 23). An increase in
this latter class of transcripts also occurs upon loss of elongation
factors ( 24). To test whether
rrp6Δ-mediated increase in
SRG1 RNA perturbs SER3 expression we
measured levels of SRG1 and SER3
mRNA by Northern blot ( B) and primer extension (see Fig. 8, which is
published as supporting information on the PNAS web site). As shown
previously ( 22), loss of Snf2p
function leads to dramatic accumulation of SER3 mRNA
and an apparent reduction in SRG1 RNA (compare lane 1
and lane 6). Loss of Rrp6p (lane 3) or Trf4p (lane 4) increases the
level of SRG1 (see also A); however,
SER3 mRNA appears unchanged. Loss of Spt5p leads to
SER3 mRNA induction, without a large increase in
SRG1 (lane 4). In the
spt5- 194,
rrp6Δ double mutant, both RNAs accumulate
(lane 5) (see also Fig. 8). These results argue that both correct
chromatin remodeling ( 22) and
active transcription elongation are required for
SRG1-mediated repression of SER3,
and that both of these requirements suggest that it is the act of
transcription that mediates repression ( 21, 22, 24, 25). This conclusion suggests that locating other
members of this class of RNAs may reveal new instances of this type of
promoter regulation. Heterogeneous RNAs Span the Promoters of
LEU4 and IMD2. To explore the possibility that additional members of this class of
RNAs span promoters of other genes, we decided to map precisely the
5’ ends of two RNAs that accumulate in the
rrp6Δ mutant. Heterogeneous small RNAs arising
from sequences containing the promoters for the LEU4
and IMD2 genes accumulate in the absence of
rrp6Δ (Table 1 and A). We detect transcripts in
wild type whose 5’ ends correspond to the reported initiation
sites for LEU4 and IMD2 ( A and
B, lanes 1). Transcripts that initiate upstream of
the normal LEU4 and IMD2 mRNAs are
readily detectable in the rrp6Δ mutant ( A and
B, lanes 2). Several IMD2 region
RNAs observed to accumulate in the rrp6Δ
mutant are detectable in wild-type cells, suggesting that they are
synthesized in the presence of Rrp6p. In addition, it appears that the
level of RNA representing the normal IMD2 mRNA start
site is reduced. We conclude that the loss of RRP6
causes stable accumulation of transcripts that initiate within the
promoters of the LEU4 and IMD2
genes, and span the promoters in the same direction as the normal mRNA
transcript. IMD2 mRNA is induced by low levels of GTP in
wild-type cells, a condition that can be created by addition of
6-azauracil to yeast cultures ( 26). To determine whether the RNAs that overlap the
IMD2 promoter are regulated in the same fashion as
the IMD2 mRNA, we treated
rrp6Δ mutant cells with 6-azauracil and
analyzed the RNA by Northern blotting ( C). The IMD2
mRNA is dramatically induced under these conditions ( C, lanes
5–8), consistent with previous observations ( 26). The levels of the small RNAs
from the IMD2 promoter are high in the
rrp6Δ mutant as shown previously ( and ) and seem to decrease
somewhat over the course of the experiment ( C). This effect may be due to
the general inhibition of transcription caused by 6-azauracil ( 27) or could reflect down-regulation
of the IMD2 promoter-associated RNA. Rrp6p is clearly
not required for the induction of IMD2 expression,
and it seems unlikely that the IMD2
promoter-associated RNAs are transacting repressors of
IMD2. We conclude that the transcription of the
IMD2 promoter-associated RNAs is regulated
differently from the IMD2 mRNA, despite apparently
arising from closely overlapping regions of the chromosome. This
arrangement is at least superficially similar to SRG1
and SER3, suggesting that regulation of the
IMD2 small RNA by guanosine levels could lead to
IMD2 induction. Heterogeneous Unstable RNAs Most Frequently Arise from Standard
mRNA Promoters Genome-Wide. Given the overlap of the small RNAs with the promoters for
LEU4 and IMD2 mRNAs, we asked
whether a high intergenic signal in the rrp6Δ
mutant is generally associated with promoter-containing intergenic
regions as compared with other intergenic regions. Reasoning that
yeast promoter elements are likely to be more common upstream of
genes, we sorted intergenic regions into one of three classes
(convergent, n = 1,601; tandem,
n = 3,018; or divergent, n
= 1,597), depending on the orientation of the two annotated
genes on either side of the intergenic region ( and Table 3, which is published as supporting
information on the PNAS web site). We expected promoters to occur more
often in divergent regions, next most often in regions between tandem
genes, and least often in regions between convergent genes. As a
group, intergenic regions situated between divergently transcribed
genes show the most increased transcription in the
rrp6Δ mutant (). Intergenic regions between tandemly
transcribed genes are moderately affected, and those situated between
convergently transcribed genes are the least affected (). To test the null hypothesis
that the difference between these distributions could be due to
chance, we applied an unpaired t test between the
convergent and divergent distributions. The likelihood
( p value) that these distributions are the same is
<0.0001. These results suggest that in the
rrp6Δ mutant, promoter-associated RNAs
distinct from true mRNAs are stabilized. In some regions not expected
to contain mRNA promoters, a similar process appears to happen (e.g.,
iYCRCΔ6; ),
suggesting that regions with sequence features that resemble promoters
may be functional but not normally detectable in wild-type cells due
to the action of Rrp6p and Trf4p (see ref. 13). We have analyzed regions of the yeast genome whose expression is
altered in the rrp6Δ mutant, as detected by
microarrays representing the complete genome (). As expected ( 28), many of the most affected genomic regions encode
known snoRNAs ( and Table 2). In the unannotated regions, we
found one new C/D box snoRNA ( and 7), as well as an unanticipated class () of
promoter-associated RNAs (– and 8) whose accumulation is
prevented by components of the Trf4p/Rrp6p decay pathway ()
( 13– 16). A New Class of RNA Transcripts with an Uncertain Relationship
to Native Gene Function. Our findings () support
the existence of a new class of heterogeneous transcripts ranging in
size from 250–800 bp in length. These RNAs are difficult to
detect convincingly in wild-type cells ( A and B);
however, deletion of RRP6 results in the strong
accumulation of this class of RNAs as polyadenylated species ( A) (see ref. 13). Because previous work used arrays that carried probes only for
previously discovered RNA transcripts ( 13), the genomic extent of such transcripts could not
be determined. Because we have used arrays covering the entire genome,
we have discovered many previously unannotated RNAs ( and Table
1). We propose that this class of unstable transcripts are produced at
normal mRNA promoters as well and in many cases can link their
association with the mRNA promoter regions of standard genes across
the genome (–7). These RNAs arise as a consequence of
RNA polymerase II activity that normally does not lead to, and may in
fact repress, the synthesis of functional mRNA ( and ) ( 21– 25).
This observation may explain in part why microarray data combined with
SVM analysis recognized an rrp6Δ phenotype
consistent with transcriptional defects, rather than decay ( 8). Relationships Between SRG1 and Promoter-Associated
RNAs. Transcription of SRG1 is required to repress
production of the SER3 mRNA by a transcriptional
interference mechanism ( 21,
22). We find that the
IMD2 and LEU4 promoters also have
promoter-associated transcripts (). However, unlike the
SRG1- SER3 system, mutations in the
TATA boxes upstream of the promoter-associated RNAs do not result in
induction of IMD2 or LEU4 mRNAs
( 29, 30). This suggests either that repression of
IMD2 and LEU4 does not require
intergenic transcription or that the intergenic transcripts and their
linked mRNAs ( LEU4 and IMD2) are
both dependent on the same core promoter elements. Despite arising
from within the promoter, the IMD2
promoter-associated RNAs do not respond to the same transcription
regulatory mechanisms as the IMD2 mRNA. For example,
the IMD2 promoter-associated RNA is robustly
transcribed under conditions in which transcription of
IMD2 mRNA is repressed ( C). Unlike
IMD2 mRNA, the IMD2
promoter-associated RNA appears to be repressed in response to 6AU
( C)
[although inhibition of overall transcription is a consequence
of lowering intracellular NTP levels ( 27), we do not yet know whether this RNA is more
inhibited than the average RNA by 6AU]. In contrast, the
IMD2 mRNA is readily induced in wild-type cells
( 31), and this does not require
Rrp6p ( C).
Given the common origin but independent regulation of these two
classes of transcripts, it is possible that this region contains two
promoters in tandem, similar to
SRG1- SER3. We suggest that the
production of unstable RNAs is a property of compound promoters, and
that in some cases they indicate the presence of an auxiliary promoter
that helps preserve a repressed or activated transcriptional state
independent of the production of mRNA. In some cases the repressed
state must be maintained by active transcription because loss of both
chromatin remodeling factors and elongation factors can lead to
inappropriate induction ()
( 21– 25). Additional details are available in Supporting
Methods, which is published as supporting information
on the PNAS web site. Yeast Strains. The following yeast strains were ordered from Open Biosystems.
Wild-type strain BY4741 ( MATa,
his3Δ 1,
leu2Δ 0,
met15Δ 0,
ura3Δ 0) was compared with
strains YSC1021-551682 ( MATa,
his3Δ 1,
leu2Δ 0,
met15Δ 0,
ura3Δ 0,
rrp6:: kanMX) and YSC1021-554220
( MATa, his3Δ 1, leu2Δ 0,
met15Δ 0,
ura3Δ 0,
trf4:: kanMX). The strain containing
the snr87 deletion, YWD452, is described in ref. 32. Strain DY884 is
described in ref. 33 and is deleted for IMD1,
IMD3, and IMD4. Strain CD4KO was
made through a cross between strain DY884 and YSC1021-551682 and is
deleted for RRP6, IMD1,
IMD3, and IMD4. Strain FY300 carries
the spt5- 194 allele
( MATa,
his4- 912Δ,
lys2- 128Δ,
leu2Δ 1,
ura3- 52,
spt5- 194). Strain GHY1344 carries
the spt5- 194 and
rrp6Δ alleles
( MATa,
lys2- 128Δ,
leu2Δ, ura3Δ,
spt5- 194,
rrp6:: KanMX). Before RNA isolation,
strains carrying the spt5- 194 allele
were heat-shifted to 39°C for 30 min. Stain GHY15 contains the
SNF2 mutant allele
( MATa,
his4- 194Δ,
his3Δ 200,
lys2- 128Δ,
ura3- 52,
trp1Δ 63,
snf2Δ 1:: HIS3). Oligonucleotides. Oligonucleotides were ordered from Sigma-Genosys in pairs designed
to amplify the part of each candidate locus except 50–100 bp
from the ends of the flanking ORFs. This approach was used where
possible to avoid hybridization to the 5’ or 3’ UTRs of
mRNAs coming from known ORFs. Sequences are available in
Supporting Methods. RNA Isolation. RNA was isolated by using a hot phenol treatment as described in
ref. 9 and treated with RQ1 DNase (Promega). Oligo(dT) affinity
chromatography was done by using Qiagen Oligotex kits. Whole-Genome Microarray Analysis. Total RNA (20 μg) was reverse-transcribed in the presence of
Cy3- or Cy5-dUTP by using a mixture of oligo(dT) and random hexamers
as described in ref. 9. The labeled cDNAs were hybridized to
whole-genome PCR microarrays (gift of J. DeRisi, University of
California, San Francisco) overnight at 62°C. Arrays were
washed and scanned by using a GenePix 4000A scanner (Axon
Instruments). Data were normalized to the average of all features on
the array. The log 2 ratios are averages
from two reverse-labeled experiments. Intergenic assignments as
convergent, divergent, or tandem were made by using the October 2003
annotation of the yeast genome and a program written by Leslie Grate
(Table 3). Array data were deposited in the Gene Expression Omnibus
under accession numbers GSE3813 and GSM86408. Northern Blots. Agarose-formaldehyde (1.5%, 2.2 M) gels were blotted onto
nylon (Hybond-N, Amersham Pharmacia) in 10× SSC (1× SSC
= 0.15 M sodium chloride/0.015 M sodium citrate, pH 7). RNA was
UV-crosslinked to the nylon by using the Stratagene Stratalinker.
Acrylamide (6%), 7 M urea gels were electrophoretically
transferred onto nylon (Hybond-N, Amersham Pharmacia) in 1× TBE
(89 mM Tris/89 mM boric acid/2.5 mM EDTA, pH 8.3) at 400 mA for 4 h
and UV-crosslinked. After transfer, membranes were stained with
methylene blue to verify loading and transfer. Membranes were
hybridized for 12 h at 42°C in 500 mM phosphate buffer,
7% (wt/vol) SDS, 1 mM EDTA (pH 8.0), and 1% (wt/vol)
BSA. About 106 cpm of probe was added to
each hybridization mix. After incubation, the membranes were washed
twice at room temp in 2× SSC and once in 0.1×
SSC/0.1% SDS at 65°C, exposed to storage phosphor
screens, and scanned with a phosphorimager for analysis. Radioactive probes were generated by denaturing 100 ng of
gel-purified PCR product in the presence of 5 pmols of primer as
indicated at 60°C for 10 min. After cooling on ice for 5 min,
extensions were carried out in 50 mM Tris·Cl (pH 7.5), 10 mM
MgCl2, 1 mM DTT, 0.05 mg/ml BSA, 5 units
of exonuclease-free Klenow, 50 μCi (1 Ci = 37 GBq) of
[α-32P]dATP, 80
μM dCTP, 80 μM dGTP, 80 μM dTTP, and 0.5
μM dATP at 37°C for 15 min. After extension,
unincorporated nucleotides were removed by passing the probes over a
G-50 Sephadex resin. Mapping Ribose Methylation by Primer Extension. Primer extension using reverse transcriptase under limiting dNTPs
to test for ribose methylation in 18S rRNA was done as described in
ref. 20. The position of the modification stop was determined by
measuring the distance from the 5’ end of the priming oligo
using a radioactive 10-bp marker. 5’-End Mapping of Promoter-Associated RNAs
IMD2 and LEU4. Ten micrograms of total RNA from the indicated strains was annealed
with 0.3 pmols of32P-kinased oligo at
60°C for 10 min and then snap-cooled on ice. After reverse
transcription [30 min at 42°C in 50 mM Tris·Cl
(pH 8.0)/60 mM NaCl/5 mM MgCl2/10 mM
DTT/0.4 mM dNTPs/50 units of SuperScript II reverse transcriptase
(Invitrogen)], the reactions were extracted,
ethanol-precipitated, and resolved on a 6% polyacrylamide, 7 M
urea gel. Induction of IMD2 by 6-Azauracil
Treatment. Yeast strain YSC1021-551682 was grown to saturation in SCD-Ura,
diluted to an OD600 of 0.8, and induced
with 6-azauracil (75 μg/ml) at 26°C for the times
indicated in . Total RNA (5 μg) from each time point was
loaded onto an agarose-formaldehyde gel, blotted, and probed with DNA
generated from amplification of chromosome 8 between positions 553716
and 554377 and Klenow extension by using 5’-gtggagttattaattgcactg as the priming oligo. We thank Joe DeRisi for providing microarrays, Tyson Clark and Karen Artiles for hybridizations in the early stages of this work, Leslie Grate for computational help, Wayne Decatur for strain YWD452, Todd Lowe for sharing his knowledge of snoRNAs, Grant Hartzog and John Tamkun for insights into transcription, and Marv Wickens for encouraging words. This work was supported by National Institutes of Health Grant GM040478. | | snRNA | small nuclear RNA | | | snoRNA | small nucleolar RNA |
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