Báo cáo khoa học: A simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesis doc

A simple in vivo assay for measuring the efficiency
of gene length-dependent processes in yeast mRNA
biogenesis
Macarena Morillo-Huesca, Manuela Vanti and Sebastia
´
n Cha
´
vez
Departamento de Gene
´
tica, Universidad de Sevilla, Seville, Spain
Gene expression is a multistep process involving tran-
scriptional and post-transcriptional events. RNA
polymerase II-dependent transcription starts by the
assembly of the pre-initiation complex (PIC), followed
by the initiation step. After initiation, transcription
elongation is coupled with a set of RNA modifications
(capping, splicing and polyadenylation) occurring
along the transcription unit. Transcription termination
is connected to the RNA cleavage required for tran-
script polyadenylation. Formation of mRNP, the
mRNA–protein complex transportable to the cyto-
plasm, is also linked to transcription elongation [1].
The traditional view of transcription and RNA pro-
cessing as separate events has been replaced by mRNA
biogenesis as a new concept involving a complex net of
functional interactions between RNA processing and
the different steps of transcription [2,3].
Some elements of the gene expression machinery
play a role at the initial steps of mRNA biogenesis
whereas some others act all along the transcription

unit. Among the former, we find the general transcrip-
tion factors involved in PIC assembly, initiation and
early elongation [4,5]. The mechanisms of transcrip-
tional regulation of gene expression that take place
during these early events have been extensively studied
and are fairly well understood [6,7]. The latter set of
elements is formed by those factors interacting with
RNA polymerase II all along transcription elongation
Keywords
gene length; mRNA biogenesis; reporter
system; Saccharomyces cerevisiae;
transcription elongation
Correspondence
S. Cha
´
vez, Departamento de Gene
´
tica,
Universidad de Sevilla, Facultad de Biologia,
Avda ⁄ Reina Mercedes, 6, E41012-Sevilla,
Spain
Fax: + 34 954557104
Tel: +34 954550920
E-mail:
Enzymes
Acid phosphatase (EC 3.1.3.2)
(Received 26 September 2005, revised
14 December 2005, accepted 16 December
2005)
doi:10.1111/j.1742-4658.2005.05108.x

We have developed a simple reporter assay useful for detection and analysis
of mutations and agents influencing mRNA biogenesis in a gene length-
dependent manner. We have shown that two transcription units sharing the
same promoter, terminator and open reading frame, but differing in the
length of their 3¢-untranslated regions, are differentially influenced by muta-
tions affecting factors that play a role in transcription elongation or RNA
processing all along the transcription units. In contrast, those mutations
impairing the initial steps of transcription, but not affecting later steps of
mRNA biogenesis, influence equally the expression of the reporters, inde-
pendently of the length of their 3¢-untranslated regions. The ratio between
the product levels of the two transcription units is an optimal parameter with
which to estimate the efficiency of gene length-dependent processes in mRNA
biogenesis. The presence of a phosphatase-encoding open reading frame in
the two transcription units makes it very easy to calculate this ratio in any
mutant or physiological condition. Interestingly, using this assay, we have
shown that mutations in components of the SAGA complex affect the level
of mRNA in a transcript length-dependent fashion, suggesting a role for
SAGA in transcription elongation. The use of this assay allows the identifica-
tion and ⁄ or characterization of new mutants and drugs affecting transcrip-
tion elongation and other related processes.
Abbreviations
GLAM, gene length-dependent accumulation of mRNA; MPA, mycophenolic acid; ORF, open reading frame; PIC, pre-initiation complex;
3¢-UTR, 3¢-untranslated regions; SAGA, Spt-Ada-Gcn5-Acetyltransferase.
756 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS
[8,9]. The relative contribution of this second kind of
element to gene expression is expected to depend on
the length of the transcription unit, it being greater in
longer genes than in shorter ones.
The distinction between these two types of elements
is not always easy when analyzing a mutant or a

physiological agent affecting gene expression. Nuclear
run-on allows measurement of transcription elongation
efficiency along the transcription unit [10], but it is time-
consuming and not easy to carry out in a high number
of samples. In addition, the information obtained by
nuclear run-on shows the location of active polymerases,
but it gives no information about the quality of the
mRNA that is being synthesized. This also happens
when RNA polymerase II location within the transcrip-
tion unit is analyzed by chromatin immunoprecipita-
tion; although, in this case, it is possible to distinguish
between those elements influencing transcription
elongation rate and those involved in processivity [11].
Other in vivo assays, such as sensitivity to 6-azauracil or
to mycophenolic acid, have been used in yeast to detect
transcription elongation defects, and they are easy to
perform [12]; however, those assays are too indirect to
obtain solid conclusions [13]. In vitro assays are useful
for defining the role of a given element in a specific
step of gene expression (e.g. transcription initiation,
transcription elongation or splicing), but they are also
time-consuming and not recommended as the first assay
with which to classify a mutant.
The best assay for a rapid evaluation of gene expres-
sion is the use of a reporter system. Available reporter
systems allow detection of defects in gene expression,
but cannot distinguish between an impairment of the
initial steps of transcription, including promoter regu-
lation, and an effect on the subsequent events of
mRNA biogenesis.

We have shown elsewhere that expression of long
transcription units in Saccharomyces cerevisiae is more
sensitive to mutations affecting the Tho2-Hpr1-Mft1-
Thp1 (THO) complex [14], connected to transcription
elongation and mRNP formation [15]. Taking the
dependence on the length of the transcription unit as a
criterion, we have developed a reporter assay useful
for detecting mutations and agents influencing mRNA
biogenesis all along the transcription unit. In this
article, we show that two transcription units sharing
the same promoter, terminator and reporter open read-
ing frame (ORF), but differing in the length of their
3¢-untranslated regions (3¢-UTR), are differently influ-
enced by mutations affecting factors that play a role in
mRNA biogenesis all along transcription elongation.
In contrast, those mutations exclusively impairing the
initial steps of transcription influence equally the
expression of the reporters, independently of the length
of their 3¢-UTR.
Results
An in vivo assay to measure gene length-
dependent efficiency of mRNA accumulation
We constructed several plasmids containing the PHO5
coding region transcribed under the control of
the GAL1 promoter, but differing in the length of their
3¢-UTR. To increase the length of the 3¢-UTR we
inserted Escherichia coli lacZ (either a short fragment
or the entire gene in both orientations) or its eukaryotic
homolog Kluyveromyces lactis LAC4. These two
sequences are equally large but differ in G + C and

chromatin organization (see Discussion). Figure 1(A)
shows the relative length of the 3¢-UTR of every tran-
scription unit used in this work.
The ability of the different transcription units to
produce full-length mRNA was tested by performing
northern analysis on cultures of the respective trans-
formants grown in a galactose-containing medium. In
all cases, a unique transcript of the expected length
was detected (Fig. 1B). The intensity of the mRNA
signals corresponding to the wild-type cells inversely
correlated to the length of the transcription units (see
lanes 1–5 in Fig. 1B). This result does not necessarily
mean that the shortest transcripts (PHO5 alone or
PHO5::lacZD) accumulated at a higher level than the
longest ones; length also might have influenced the
yield of mRNA extraction from the cells and the effi-
ciency of transference during blotting. This is likely to
be the reason for the variability observed when the
results of independent northern experiments are com-
pared (see below). In any case, the quantification of
the northern blots indicated that the three longest
transcription units showed very similar levels of
mRNA accumulation (Fig. 1B), despite the sequence
of its 3¢-UTR being different (E. coli lacZ in both ori-
entations or K. lactis LAC4).
We hypothesized that those mutations affecting
transcription elongation all along the transcription unit
should produce a negative length-effect on mRNA
accumulation, which was stronger than the one that
the wild type might exhibit. In order to test this hypo-

thesis, we chose the spt6–140 allele, a thermosensitive
mutation affecting a bona fide transcription-elongation
factor that colocalizes with RNA polymerase II along
the transcription unit [16–18]. As shown in Fig. 1(B),
even at permissive temperature, the signals of the three
long transcripts were severely reduced in the mutant
strain compared with the wild type, whereas the signals
M. Morillo-Huesca et al. Assay for gene length-dependent mRNA biogenesis
FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 757
of the short transcripts suffered milder effects. To bet-
ter estimate the effect of length, we calculated the
ratios between the mRNA signals of a strain expres-
sing the long transcription units vs. the mRNA signal
of the same strain expressing just PHO5 (Fig. 1C).
This long- ⁄ short-transcript ratio was clearly lower in
spt6–140 than in the wild type, even when the mRNA
containing the 0.3 kb fragment of lacZ was considered
the ‘long’ one. However, this ratio was dramatically
reduced in the mutant when applied to the longest
transcription units, being around 5 times lower than in
the wild type (Fig. 1C).
PHO5 encodes a periplasmic acid phosphatase,
which is very easy to assay [19]. The levels of acid
phosphatase activity of an untransformed S. cerevisiae
strain in SC-galactose medium are almost undetecta-
ble, as the endogenous PHO5 gene is repressed in
media containing high levels of phosphate [20]. As
expected, the levels of phosphatase activity in strains
lacking our reporter systems were extremely low (not
shown). Therefore, the levels of acid phosphatase

expressed by our transformants should reflect the
amounts of their respective plasmid-encoded PHO5
mRNAs accumulated in the cell. Nevertheless, in order
A
B
C
D
Fig. 1. Influence of gene-length on mRNA accumulation. (A) Transcription units used in this work, corresponding to plasmids pSCh202,
pSCh212–18, pSCh212, pSCh211 and pSCh209-LAC4. (B) Northern blot showing the mRNA levels of the five transcription units described in
(A) in a wild-type (MMY5.1) and an isogenic spt6–140 strain (MMY5.2). Values were normalized with respect to 25S rRNA. Three independ-
ent experiments were averaged. (C) Relative values of the mRNA levels shown in (B) with respect to the shortest transcript (PHO5 mRNA).
(D) GLAM ratios of congenic wild-type and spt6–140 strains: relative levels of acid phosphatase activity expressed by the indicated transcrip-
tion unit, with respect to the acid phosphatase activity from the shortest transcription unit (PHO5). Averages of four wild-type strains
(MMY5.1 ⁄ 4 ⁄ 6 ⁄ 7) and four spt6–140 strains (MMY5.2 ⁄ 3 ⁄ 5 ⁄ 8) are shown. For each strain, the average of at least three experiments was
considered. Error bars indicate standard errors.
Assay for gene length-dependent mRNA biogenesis M. Morillo-Huesca et al.
758 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS
to reduce the error caused by the residual expression
of the endogenous PHO5, we subtracted the residual
acid phosphatase activities of the untransformed
strains to all the following results (see Experimental
procedures). We assayed acid phosphatase activity in
eight congenic strains transformed with our five plas-
mids: four of the strains being wild-type for SPT6 and
four of them having a spt6–140 allele. The average of
the acid phosphatase activities was used to calculate
the ratios between those cells expressing the long tran-
scripts and those expressing the minimal PHO5
mRNA. Figure 1(D) shows the calculated ratios. When
we focused on the wild type, the comparison of the

acid phosphatase ratio calculated for PHO5::lacZD to
the ratios calculated for the three longest transcription
units did not indicate a significant influence of length
on Pho5 accumulation. As previously suspected, this
result suggests that the apparent effect of length on
mRNA accumulation in the wild type may be the con-
sequence of a technical bias against long mRNAs
during northern experiments. Nevertheless, and in
agreement with the mRNA ratios shown in Fig. 1(C),
the acid phosphatase ratios of the spt6 mutant were
again clearly lower than those corresponding to the
wild type, with the difference being stronger when the
long transcript corresponded to any of the three lon-
gest 3¢-UTRs (Fig. 1D). Moreover, the absolute values
of the acid phosphatase ratios are highly reproducible,
whereas the mRNA ratios are consistent within a sin-
gle northern experiment, but show a high variability
when comparing different experiments, probably due
to differences in mRNA extraction and ⁄ or mRNA
transfer during blotting. We concluded that measure-
ment of acid phosphatase activity was the best estima-
tion of the mRNA abundance in our systems, and
from here on we use the ratio of acid phosphatase
activities as an indicator of gene length-dependent
accumulation of mRNA (GLAM).
Reduced GLAM ratios in transcription-elongation
mutants
To further confirm that the GLAM ratio is a valid
parameter with which to detect defects in mRNA bio-
genesis, we extended this assay to a set of well-known

mutants affected in transcription elongation.
Three spt16–197 strains, affected in one of the sub-
units of the yFACT complex [21], clearly showed
lower GLAM ratios than three congenic wild types
(Fig. 2A). The mRNA ratios of a spt16–197 strain and
a congenic wild type confirmed the reliability of the
phosphatase results (Fig. 2B). A similar pattern of
GLAM ratios was obtained when comparing an spt4D
mutant [17,22], lacking one of the subunits of the yD-
SIF complex, with an isogenic wild type (Fig. 2C).
Since the length effect was especially clear when using
the transcription units containing the longest 3¢-UTR,
we chose GAL1pr::PHO5-lacZ and GAL1pr::PHO5-
LAC4 for the following assays. The mRNA ratios cal-
culated for these two transcription units in the spt4D
strain and in the isogenic wild type confirmed again
the validity of the GLAM ratio to predict elongation
defects (Fig. 2D).
Four additional mutants affected in transcription
elongation showed decreased GLAM ratios compared
with an isogenic wild type: rpb9D, a 6-azauracil-sensi-
tive mutant lacking a subunit of RNA polymerase II
[23]; leo1D and rtf1D, two mutants lacking subunits of
the PAF-complex [24,25]; and elp3D, a mutant lacking
the histone acetyltransferase subunit of the elongator
[26] (Fig. 2C). In all these cases, the GLAM ratios of
the mutants were, at most, 50% of the wild-type ratios
and, therefore, we consider this percentage as the
threshold value to indicate a deficiency in gene length-
dependent mRNA biogenesis.

TFIIS, encoded by the DST1 ⁄ PPR2 gene in S. cere-
visiae, is a very well known transcription elongation
factor involved in releasing RNA polymerase II from
arrest sites by stimulating RNA cleavage [27].
Although the role of TFIIS in elongation has always
been connected to specific pause-sites, and it has never
been shown to play a general role in RNA polymerase
II-dependent transcription, we performed our assay in
a dst1D mutant. The GLAM ratios in a dst1D mutant
did not show a significant difference with respect to an
isogenic wild-type strain when GAL1pr::PHO5-LAC4
was considered the long transcription unit, and only
showed a weak decrease when GAL1pr::PHO5-lacZ
was chosen (Fig. 3A). We repeated the assays in the
presence of sublethal doses of the NTP-depleting drug
mycophenolic acid (MPA), a drug that has been repor-
ted to enhance the transcriptional defects of dst1D
mutants by reducing RNA polymerase II processivity
[11]. As is expected for an inhibitor of transcription
elongation, the presence of 2 lgÆmL
)1
MPA reduced
the GLAM ratio of the wild-type strain (Fig. 3A).
Similar results were obtained with 6-azauracil (not
shown), a drug that also causes depletion of the NTP
pools, confirming the suitability of this assay for ana-
lyzing elongation-inhibiting drugs. However, MPA,
even at 10 lgÆmL
)1
, did not decrease further the

GLAM ratio of the dst1D mutant (Fig. 3A).
As these results separate TFIIS from the other elon-
gation factors studied in this work, we performed nor-
thern blot experiments to confirm this negative result
based on acid phosphatase data. Figure 3(B) shows a
M. Morillo-Huesca et al. Assay for gene length-dependent mRNA biogenesis
FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 759
representative northern experiment which illustrates
the absence of a dst1D effect on GLAM; neither
the PHO5-lacZ ⁄ PHO5 mRNA nor the PHO5-LAC4 ⁄
PHO5 ratio was affected by dst1D. Moreover, the pres-
ence of MPA significantly reduced the mRNA levels
present in dst1D, but the effect on the long transcrip-
tion units was proportionally similar to the effect
caused on the minimal PHO5 mRNA, therefore pro-
ducing similar ratios in the wild type and in the
mutant strain (Fig. 3C). Our results confirm that
TFIIS is not playing a general role in mRNA biogen-
esis all along the transcription unit.
GLAM ratios are not affected by the impairment
of PIC assembly or transcription initiation
With the exception of dst1D, all transcription elonga-
tion mutants assayed so far showed reduced GLAM
ratios. One possible explanation for these results is an
indirect effect on the GLAM ratios of any major
impairment of transcription, regardless of the step of
mRNA biogenesis where it occurs. To rule out this
possibility, we assayed a wide variety of mutants affec-
ted in PIC assembly and transcription initiation. We
analyzed a TBP mutant [28]; a mot1 mutant, affected

A
B
C
D
E
Fig. 2. Mutants affected in transcription elongation factors show reduced GLAM ratios. (A) Averaged GLAM ratios (see legend of Fig. 1) of
three wild types (MMY11.3 ⁄ 8 ⁄ 12) and three congenic spt16–197 strains (MMY11.4 ⁄ 6 ⁄ 10). (B) Northern blot and mRNA-ratios (see legend
of Fig. 1) of a wild-type (MMY11.3) and a congenic spt16–197 strain (MMY11.4). Notation of transcription units as in Fig. 1(A). (C) GLAM
ratios of the spt4D strain Y06986 and the isogenic wild-type BY4741. (D) Northern blot and mRNA ratios of Y06986 and BY4741. E. GLAM
ratios of rpb9D, leo1D, rtf1D, elp3D and an isogenic wild type (strains Y04437, Y02379, Y04611, Y02742 and BY4741).
Assay for gene length-dependent mRNA biogenesis M. Morillo-Huesca et al.
760 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS
in one of the main TBP regulators [29]; a toa1 mutant,
affected in the large subunit of yTFIIA [29]; two sua7
mutants, affected in yTFIIB [30]; two tfa1 mutants,
affected in the large subunit of yTFIIE [31]; and two
mutants, srb10D and srb11D, lacking subunits of the
cyclin–kinase complex that negatively regulates tran-
scription initiation [32,33]. None of them showed a sig-
nificant effect on the GLAM ratios, when compared
with the isogenic wild-type strains (Fig. 4A–E). Only
toa1–18 showed a slightly reduced GLAM ratio when
PHO5-lacZ was used as the long transcript, but not
when PHO5-LAC4 was considered, excluding a general
effect of toa1–18 on GLAM (Fig. 4B). Taking together
all the results shown in Fig. 4, we concluded that the
GLAM ratios are not affected by alterations of PIC
assembly or transcription initiation.
Modification of the chromatin structure is an
important requirement for transcription regulation and

PIC assembly at many promoters. One of the main
factors involved in this process is the SAGA complex
[34], up to now mainly connected to the initial steps of
transcription. We assayed our reporters in two mutants
affecting subunits of SAGA. Both gcn5D, a mutant
lacking the histone acetyltransferase present in SAGA,
and spt3D showed reduced GLAM ratios (Fig. 5A). As
this result suggests a role of SAGA in mRNA biogen-
esis all along the transcription unit, we performed nor-
thern blot experiments to confirm the acid phosphatase
data. As shown in Fig. 5(B), the accumulation of long
mRNAs was more sensitive to the gcn5D mutation
than the accumulation of the minimal PHO5 transcript
rendering, thus significantly lower ratios.
One possible explanation for these results is an indi-
rect effect on the GLAM ratios by any mutation gen-
erating abnormal chromatin structures. In order to test
this hypothesis, we analyzed a wide set of mutants
including deletions of histone genes like hta1htb1D [35]
or htz1D [36]; mutants affected in nucleosome remode-
ling like isw1D [37], chd1D [38] or swr1D [39]; and an
rpd3D mutant, lacking the main histone deacetylase
involved in transcription [40]. No significant decrease
of the GLAM ratios were observed in any mutant,
A
B
C
Fig. 3. dst1D, even in the presence of mycophenolic acid, does not
show reduced GLAM ratios. (A) GLAM ratios (see legend of Fig. 1)
of dst1D and an isogenic DST1 (strains MMY9.2 and BY4741) cul-

tured in the absence or in the presence of sublethal amounts of
MPA. (B) mRNA levels of the transcription units and strains ana-
lyzed in (A) cultured in the absence of MPA. The relative values of
the mRNA levels with respect to the shortest transcript (PHO5
mRNA) are also shown. Three independent experiments were aver-
aged. Notation of transcription units as in Fig. 1(A). (C) mRNA lev-
els of the transcription units and strains analyzed in (A) cultured in
the presence of MPA (10 lgÆmL
)1
). A representative experiment is
shown.
M. Morillo-Huesca et al. Assay for gene length-dependent mRNA biogenesis
FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 761
suggesting that the detected effect of the SAGA
mutants on the GLAM ratio would not be due to an
indirect influence of altered chromatin structure but to
a sustained role of SAGA after the initial steps of
transcription (Fig. 5C–D).
Influence of mRNA processing on the GLAM
ratios
Transcription elongation is coupled with mRNA pro-
cessing, since all RNA modifications leading to pro-
duce a mature exportable mRNA take place or start
during elongation. We decided to analyze a set of
mutations affecting proteins that play a post-transcrip-
tional role in gene expression. We included mft1D and
thp2D, two mutants lacking subunits of the THO com-
plex [41]; ref2D and syc1D, involved in the 3¢ cleavage
previous to polyadenylation and termination [42,43];
and three mutants, cdc40D, cus2D and cwc15D, lacking

proteins connected to RNA splicing [44–46]. As expec-
ted from the reported requirement of the THO com-
plex for the expression of long genes [14], the GLAM
ratios of the two THO mutants were dramatically
reduced when compared with the wild type (Fig. 6).
The absence of effect of cdc40D and cus2D on the
GLAM ratios was also expected, as no intron is
located in the transcription units used in this study.
However, cwc15D, another mutant connected to spli-
cing, did show a significant reduction (Fig. 6). ref2D
also showed low GLAM ratios, whereas syc1D,
another mutant connected to 3¢ cleavage did not
(Fig. 6). We conclude that only a subset of mRNA-
processing functions influences mRNA biogenesis in a
gene length-dependent manner.
Discussion
Some factors required for mRNA biogenesis play their
role during PIC assembly, transcription initiation and
early elongation, whereas some others functionally
interact with Pol II all along transcription elongation.
A
B
C
D
E
Fig. 4. Mutants affected in PIC assembly and transcription initiation
do not show reduced GLAM ratios. GLAM ratios (see legend of
Fig. 1) of the following mutants and their corresponding wild types:
(A) tbp1-P65S and an isogenic TBP1 (strains YAK293 and YAK289).
(B) mot1–1, toa1–18 and a wild type with the same genetic back-

ground (strains FY1214, JMY498 and FY98). (C) sua7-L50D, sua7-
K205E and an isogenic SUA7 (strains FP177, FP207 and FP142).
(D) tfa1-T218D, tfa1-C127F and an isogenic TFA1 (strains YSB326,
YSB331 and YSB324). (E) srb10D, srb11D and an isogenic wild type
(strains SLY7, SLY107 and SLY3).
Assay for gene length-dependent mRNA biogenesis M. Morillo-Huesca et al.
762 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS
In order to develop an easy test for detecting muta-
tions or drugs that influence mRNA biogenesis all
along the transcription unit, we designed a novel
two-reporter assay based on the PHO5 gene. We hypo-
thesized that gene length is the key element that distin-
guishes between factors involved in the initial steps of
transcription and factors influencing mRNA biogenesis
all along the transcription unit, as it is well established
for the well-known elongation factor ELL in Dro-
sophila cells [47]. We supposed that long transcription
units would be more strongly impaired by mutations
affecting this second kind of factors than shorter ones,
whereas those mutations causing dysfunction of a gen-
eral factor only involved in the early steps of transcrip-
tion would affect equally all transcription units,
regardless of their length. In order to quantify the
results of the assay, we have defined gene length-
dependent efficiency of mRNA accumulation as the
levels of a long mRNA encoding PHO5, divided by
the levels of the minimal PHO5 mRNA, when both
A
B
C

D
Fig. 5. Mutants affected in SAGA, but not other chromatin-related
mutants, show reduced GLAM ratios. (A) GLAM ratios (see legend
of Fig. 1) of gcn5D, spt3D and an isogenic wild type (strains
Y07285, Y04228 and BY4741). (B) Northern blot showing the
mRNA levels of the transcription units and strains (gcn5D and wild
type) analyzed in (A). The relative values of the mRNA levels with
respect to the shortest transcript (PHO5 mRNA) are also shown.
Notation of transcription units as in Fig. 1(A). (C) GLAM ratios of
hta1htb1D and an isogenic wild type (strains FY710 and FY120).
(D) GLAM ratios of htz1D, swr1D, isw1D, chd1D, rpd3D and an
isogenic wild type (strains Y01703, Y03693, Y03385, Y06160,
Y01114 and BY4741).
Fig. 6. Influence of mutations affecting mRNA processing on the
GLAM ratios. GLAM ratios (see legend of Fig. 1) of mft1D, thp2D,
cdc40D, cus2D, cwc15D, ref2D, syc1D and an isogenic wild type
(strains Y00508, Y02861, Y04201, Y01158, Y03521, Y03554,
Y02435 and BY4741).
M. Morillo-Huesca et al. Assay for gene length-dependent mRNA biogenesis
FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 763
are transcribed from the same promoter. We have
shown that the so-defined ratio can be estimated by
assaying the acid phosphatase activity encoded by
PHO5 (GLAM ratio), and that the ratios obtained
from acid phosphatase assays are in fact more repro-
ducible than those directly calculated from northern
experiments. As all transcription units used in this
assay express an identical Pho5 protein, it is unlikely
that mutations or drugs affecting translation, protein
stability or other post-translational processes, may

influence the GLAM ratios.
We tested the consistency of the GLAM ratios by
performing the assay in a wide set of previously char-
acterized mutants involved in mRNA biogenesis. The
GLAM ratios were always calculated for two different
long transcription units: the one containing E. coli
lacZ (GAL1pr::PHO5-lacZ) and the other containing
K. lactis LAC4 (GAL1pr::PHO5-LAC4 ). These two
genes share the same length, but display a very differ-
ent G + C content. Their chromatin structure in
S. cerevisiae is also completely different: random nucleo-
some positioning in lacZ [14] but translationally posi-
tioned nucleosomes in LAC4 (S. Jimeno-Gonza
´
lez,
P. M. Alepuz and S. Cha
´
vez, unpublished). We consid-
ered that a mutant showing similar GLAM ratios with
both long transcription units would indicate a gene
length-dependent effect, whereas a mutant exhibiting
differences between the GLAM ratios calculated with
each long transcript might involve sequence-dependent
or chromatin-dependent phenomena. Among all the
mutants analyzed in this study, only toa1–18 affecting
TFIIA showed a significant difference between the
GLAM ratio calculated with GAL1pr::PHO5-LAC4
and the one calculated with GAL1pr::PHO5-lacZ.We
did not find an explanation for this result, unless a
connection exists between TFIIA and the chromatin

organization of the transcribed region. In all other
strains tested, the two values were not significantly dif-
ferent, although in most cases the GLAM ratios calcu-
lated with GAL1pr::PHO5-lacZ were slightly higher
than those calculated with GAL1pr::PHO5-LAC4.
Mutations affecting transcription elongation factors,
such as SPt6, yFACT, yDSIF, and the PAF1 complex,
as well as the elongator, clearly showed lower GLAM
ratios than their isogenic wild types. In contrast, those
mutations described to affect factors involved in PIC
assembly or transcription initiation, like TBP, Mot1,
TFIIB, TFIIE or Srb10-Srb11, show very similar
GLAM ratios compared with their corresponding
wild-types, with SAGA mutations being the only
exception (discussed below). In all these mutants, the
resulting GLAM ratios for two long transcription units
(GAL1pr::PHO5-lacZ and GAL1pr::PHO5-LAC4)
were very similar. Moreover, the presence of sublethal
doses of nucleotide-depleting drugs affecting transcrip-
tion elongation also reduced the GLAM ratios. We
conclude that these general results are solid enough to
validate the novel two-reporter assay as a useful tool
with which to detect transcription elongation defects.
Mutations or drugs can affect two different aspects
of transcription elongation: elongation rate and proces-
sivity. A recent study by Mason and Struhl [11] has
shown that none of the many putative elongation fac-
tors that they tested affect the elongation rate,
although mutations in the THO complex and Spt4 sig-
nificantly reduce processivity. Our assay is based on

the comparison of transcription units of different
length, which makes it an optimal method with which
to detect processivity defects; consequently thp2, mft1D
and spt4D show the lowest GLAM ratios. Mason and
Struhl also showed that those elements affecting elon-
gation rate, such as 6-azauracil and MPA, simulta-
neously reduce processivity [11]; the decrease of the
GLAM ratios in response to these two NTP-depleting
drugs indicates that our in vivo assay can detect all the
elongation defects detected by RNA polymerase II-
dependent chromatin immunoprecipitation. Moreover,
some putative elongation mutants that did not show
reduced processivity in the study by Mason and Struhl,
such as elp3D, rtf1D and leo1D [11], did show signifi-
cantly low GLAM ratios, suggesting that our in vivo
assay displays a high sensitivity to detect elongation
defects. Finally, mutations in SPt6 and SPt16 that can-
not be analyzed by RNA polymerase II-dependent
chromatin immunoprecipitation due to technical limi-
tations of that assay [11], show reduced GLAM ratios,
supporting a contribution of FACT and SPt6 to proc-
essivity. We conclude that the new in vivo assay des-
cribed in this study is a convenient complementary
tool with which to analyze transcription elongation.
The only transcription elongation factor tested
whose mutation did not produce lower GLAM ratios
than its isogenic wild type was TFIIS. When either cal-
culated with the assayed acid phosphatase activities or
inferred from northern experiments, the ratios of a
dst1D mutant were not significantly low. Moreover,

the presence of sublethal doses of nucleotide-depleting
drugs, like 6-azauracil or MPA, reduced the GLAM
ratios of a dst1D mutant; however, it also reduced the
GLAM ratios of an isogenic wild type accordingly.
This differentiated behavior of dst1D separates TFIIS
from the other transcription elongation factors tested
in this work. The most logical explanation for this
phenomenon is that, as has been recently suggested
[11], TFIIS does not play a relevant role in elongation
all along the transcription unit, or at least along the
Assay for gene length-dependent mRNA biogenesis M. Morillo-Huesca et al.
764 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS
transcription units used in this assay. Alternately,
TFIIS may play a general function, but only during
early elongation. If this were the case, both long and
short transcription units would be equally influenced
by the absence of TFIIS, and as a result the GLAM
ratio would be unaffected. A role of TFIIS centered in
early elongation has been suggested by the genetic and
physical interactions of TFIIS [48,49]. A relevant role
of TFIIS in early elongation has been also found in
Drosophila heat-shock genes [50]. Moreover, a role of
TFIIS in activating the GAL1 promoter has been
recently demonstrated [51]. An elongation role of
TFIIS in vivo, in positions far from the promoter, has
only been shown when an artificial arrest site was
introduced, and even in this case the influence of
TFIIS depended on the degree of transcriptional acti-
vation [52]. The present results confirm the difficulties
involved in studying the dst1D mutant in vivo,an

observation already reported elsewhere [11].
The SAGA complex is one of the main elements
that mediate in activation of gene expression; it acts
through its ability to interact with gene-specific factors
and to stimulate PIC assembly. Surprisingly, gcn5D
and spt3D mutants, both lacking subunits of SAGA,
show reduced GLAM ratios. We consider it unlikely
that these gene length-dependent effects of the SAGA
mutants take place at the level of PIC assembly, since
all transcription units used to calculate the GLAM
ratios share the same promoter. We have also ruled
out an indirect effect of SAGA mutations on the
GLAM ratios provoked by overall effects on chroma-
tin structure, just as other mutations affecting struc-
tural elements of chromatin (hta1htb1D, htz1D)or
chromatin remodeling (swr1D, isw1D, chd1D, rpd3D)
show similar GLAM ratios to their isogenic wild types.
We conclude that SAGA might play an additional role
after transcription initiation all along the transcription
unit. The genetic interactions of GCN5 with the elon-
gator [53], the sensitivity of several SAGA mutants to
mycophenolic acid [54] and the genetic interactions
between genes encoding SAGA subunits and elonga-
tion factors [55,56] also suggest a role for SAGA dur-
ing transcription elongation. Alternatively, the absence
of SAGA might affect the recruitment of other factors
required for postinitiation events in mRNA biogenesis.
As discussed above, mutants affected in factors
required for transcription elongation all along the
transcription unit show reduced GLAM ratios.

However, transcription elongation is not the only
gene-length dependent process in mRNA biogenesis.
Formation of the mRNP complex, mRNA export or
splicing are other events that may be gene length-
dependent. Mutants lacking subunits of the THO
complex, involved in mRNP formation, show the
lowest GLAM ratios measured in this work. This is
not the case in other RNA processing mutants that
have been analyzed in this work. In agreement with
our results, an important part of the RNA-process-
ing machinery does not significantly influence mRNA
accumulation [15]. However, a part of the RNA pro-
cessing machinery physically interacts with elongating
Pol II; as a consequence, the absence of elements
involved in RNA processing may indirectly affect
transcription elongation [57]. This might be the
explanation of the low GLAM ratios shown by
cwc15D and ref2D, lacking proteins connected to
splicing and 3¢ cleavage, respectively. In contrast to
the consistency of the results obtained using our
reporter assay with well-known mutants affected in
transcription initiation or elongation, the behavior of
mutations affecting mRNA processing is heterogene-
ous. Additional analyses are required to understand
more fully the effect of these mutations on gene
length-dependent accumulation of mRNA.
Experimental procedures
Materials
Suppliers are indicated below at first mention, except
for chemical reagents, which were purchased from Sigma

(St. Louis, MO, USA).
Yeast strains, plasmids and media
Yeast strains used are described in Table 1. All MMY
strains were constructed by standard genetic methods of
tetrad analysis or transformation [58]. MMY5 strains are
congenic and were generated by crossing FY120 and
FY137. AGY1–10 A strain was obtained by crossing
FY348 and W303-ZT; further crossing of FY120 and
AGY-10A rendered all MMY11 strains. MMY9.2 was
obtained by sporulating Y24411.
All plasmids used are mono-copy CEN-based and are lis-
ted in Table 2. Cells were grown in yeast extract–peptone
medium or in synthetic complete medium (DIFCO, Detroit,
MI, USA), with 2% glucose or 2% galactose, at 30 °C [58].
Acid phosphatase assays
Yeast cells with the appropriated plasmids were grown on
selective synthetic medium lacking uracil with 2% galactose
and collected when cultures reached an optical density at
600 nm (OD 600) of 0.8–1. Acid phosphatase activity of
intact cells was assayed as described [19]. The acid phos-
phatase activities of the transformants were corrected by
M. Morillo-Huesca et al. Assay for gene length-dependent mRNA biogenesis
FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS 765
Table 1. Strains.
Strain Genotype Source
FP142 MATa ura3–52 trp1D63 sua7D1[pRS314 ⁄ SUA7] [30]
FP177 MATa ura3–52 trp1D63 sua7D1[pRS314 ⁄ sua7-L50D] [30]
FP207 MATa ura3–52 trp1D63 sua7D1[pRS314 ⁄ sua7-K205E] [30]
FY120 MATa leu2D1 ura3 his4–912d lys2–128d [17]
FY1214 MATa leu2D1 ura3–52 mot1–1 [29]

FY137 MATa ura3 his4–912d lys2–128d spt6–139 [17]
FY348 MATa leu2D1 ura3 his4–912d lys2–128d spt16–196 [59]
FY710 MATa leu2D1 ura3 his4–912d lys2–128d hta1-htb1::LEU1 [35]
FY98 MATa leu2D1 ura3–52 [29]
JMY498 MATa ura3–52 his4–912d lys2–128d toa1–18GSG [29]
SLY107 MATa his3D200 leu2–3,112,ura3–52 srb11D1::hisG [32]
SLY3 MATa his3D200 leu2–3,112,ura3–52 [32]
SLY7 MATa his3D200 leu2–3,112,ura3–52 srb10D1::hisG [32]
W303-ZT MATa leu2 his3 ade2 trp1 ura3::GAL-lacZ::URA2 J. Svejstrup laboratory
YAK289 MATa ura3–52 trp1–63 leu2,3–112 his3–609Dspt15[pTM8 ⁄ TBP] [28]
YAK293 MATa ura3–52 trp1–63 leu2,3–112 his3–609Dspt15[pTM1228 ⁄ TBP-P65S] [28]
YSB324 MATa ura3–52 leu2–3112 his3D200 tfa1D1::HIS3 [pNK6 ⁄ TFA1] [31]
YSB326 MATa ura3–52 leu2–3112 his3D200 tfa1D1::HIS3 [pNK1DSpe] [31]
YSB331 MATa ura3–52 leu2–3112 his3D200 tfa1D1::HIS3 [pNK6 ⁄ tfa1-C127F] [31]
BY4741 MATa his3D1 leu2D0 met15D0 ura3D0 EUROSCARF
Y00508 MATa his3D1 leu2D0 met15D0 ura3D0 mtf1::KAN EUROSCARF
Y01114 MATa his3D1 leu2D0 met15D0 ura3D0 rpd3::KAN EUROSCARF
Y01158 MATa his3D1 leu2D0 met15D0 ura3D0cls2::KAN EUROSCARF
Y01586 MATa his3D1 leu2D0 met15D0 ura3D0 snf2::KAN EUROSCARF
Y01703 MATa his3D1 leu2D0 met15D0 ura3D0 htz1::KAN EUROSCARF
Y02379 MATa his3D1 leu2D0 met15D0 ura3D0 leo1::KAN EUROSCARF
Y02435 MATa his3D1 leu2D0 met15D0 ura3D0 syc1::KAN EUROSCARF
Y02742 MATa his3D1 leu2D0 met15D0 ura3D0 elp3::KAN EUROSCARF
Y02861 MATa his3D1 leu2D0 met15D0 ura3D0 thp2::KAN EUROSCARF
Y03385 MATa his3D1 leu2D0 met15D0 ura3D0 isw1::KAN EUROSCARF
Y03521 MATa his3D1 leu2D0 met15D0 ura3D0 cwc15::KAN EUROSCARF
Y03554 MATa his3D1 leu2D0 met15D0 ura3D0 ref2::KAN EUROSCARF
Y03693 MATa his3D1 leu2D0 met15D0 ura3D0 swr1::KAN EUROSCARF
Y04201 MATa his3D1 leu2D0 met15D0 ura3D0 cdc40::KAN EUROSCARF
Y04228 MATa his3D1 leu2D0 met15D0 ura3D0 spt3::KAN EUROSCARF

Y04437 MATa his3D1 leu2D0 met15D0 ura3D0 rpb9::KAN EUROSCARF
Y04611 MATa his3D1 leu2D0 met15D0 ura3D0 rtf1::KAN EUROSCARF
Y06160 MATa his3D1 leu2D0 met15D0 ura3D0 chd1::KAN EUROSCARF
Y06986 MATa his3D1 leu2D0 met15D0 ura3D0 spt4::KAN EUROSCARF
Y07285 MATa his3D1 leu2D0 met15D0 ura3D0 gcn5::KAN EUROSCARF
Y24411 MATa ⁄ a his3D1 ⁄ his3D1leu2D0leu)2D0 lys2D0 ⁄ LYS2 MET15 ⁄ met15D0 ura3D0 ⁄ ura3D0 dst1::KAN ⁄ DST1 EUROSCARF
AGY1–10A MATa ade2 trp1leu2 his4–912d lys2–128d ura3::GAL1-lacZ::URA3 spt16–197 This study
MMY5.1 MATa ura3 his4–912d lys2–128d This study
MMY5.2 MATa ura3 his4–912d lys2–128d spt6–140 This study
MMY5.3 MATa ura3 his4–912d lys2–128d leu2 spt6–140 This study
MMY5.4 MATa ura3 his4–912d lys2–128d leu2 This study
MMY5.5 MATa ura3 his4–912d lys2–128d spt6–140 This study
MMY5.6 MATa ura3 his4–912d lys2–128d This study
MMY5.7 MATa ura3 his4–912d lys2–128d leu2 This study
MMY5.8 MATa ura3 his4–912d lys2–128d leu2 spt6–140 This study
MMY9.2 MATa his3D1 leu2D0 met15D0 ura3D0 dst1::KAN This study
MMY11.3 MATa trp1leu2 his4–912d lys2–128d ura3 This study
MMY11.4 MATa ade2 trp1 his4–912d lys2–128d ura3 spt16–197 This study
MMY11.6 MATa ade2 leu2 his4–912d lys2–128d ura3 spt16–197 This study
MMY11.8 MATa ade2 leu2 his4–912d lys2–128d ura3 This study
MMY11.10 MATa trp1leu2 his4–912d lys2–128d ura3 spt16–197 This study
MMY11.12 MATa leu2 his4–912d lys2–128d ura3 This study
Assay for gene length-dependent mRNA biogenesis M. Morillo-Huesca et al.
766 FEBS Journal 273 (2006) 756–769 ª 2006 The Authors Journal compilation ª 2006 FEBS
subtracting the residual acid phosphatase activity of the
corresponding untransformed strain.
Northern analyses
Six micrograms of total RNA prepared from yeast cells,
collected in the conditions described above, were subjected
to electrophoresis on formaldehyde-agarose gels (agarose

form Pronadisa, Madrid, Spain), transferred to Hybond-N
filters (Amersham Biosciences UK, Buckinghamshire, UK),
and UV cross-linked prior to hybridization at 65 °Cin
0.5 m sodium phosphate buffer, pH 7, 7% SDS, with a
[
32
P]dCTP-labeled DNA PHO5 probe. Quantification of
mRNA levels was performed in a Phosphorimager. All val-
ues were normalized with respect to the present amount of
25S rRNA, detected by hybridization with a [
32
P]-oligo-
labeled 589 bp rDNA internal PCR fragment amplified
with the 19-mer oligonucleotides 5¢-TTGGAGAGGGCA
ACTTTGG-3¢ and 5¢-CAGGATCGGTCGATTGTGC-3¢
(Stab Vida, Oeiras, Portugal).
Acknowledgements
We thank Francisco Malago
´
n and Francisco Navarro
for their critical reading of the draft; we also thank
S. Buratowski, T. Kokubo, A. Ponticelli, J. Svejstrup,
F. Winston and R. Young for strains and plasmids,
and Antonio Garcı
´
a-Susperregui for strains construc-
tion. This work was supported by the Ministry of
Education and Science of Spain (grant BMC2003-
07072-C03-01 to SC and fellowships to MM-H and to
MV), and by the Andalusian Government (CVI271).

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