Sequence requirements of the bidirectional yeast
TRP4
mRNA 3
'-end formation signal
Sequence requirements of the bidirectional yeast TRP4 mRNA 3 '-end formation signal
Christoph M.
Egli
,
Katrin
Düvel
,
Nathalie
Trabesinger-Rüf
+
,
Stefan
Irniger
and
Gerhard H.
Braus*
Institute of Microbiology, Georg-August University, Grisebachstraße 8, D-37077
Göttingen
,
Germany
Received July 15, 1996;
Revised and Accepted November 13, 1996
ABSTRACT
The yeast
TRP4
3
'
-end formation signal functions in both orientations in an
in vivo
test system. We show here that the
TRP4
3
'
-end formation element consists of two functionally different sequence
regions. One region of
~
70 nucleotides is located in the untranslated region between the translational
stop codon and the major poly(A) site. The major poly(A) site is not part of
this region and can be deleted without a decrease in
TRP4
3
'
-end formation. 5
'
and 3
'
deletions and point mutations within this region affected 3
'
-end formation similarly in both orientations. In the center of this region
the motif TAGT is located on the antisense strand. Point mutations within this
motif resulted in a drastic reduce of 3
'
-end formation activity in both orientations. A second region consists of
the 3
'
-end of the
TRP4
open reading frame and is required for 3
'
-end formation in forward orientation. A single point mutation in a TAGT motif of the
TRP4
open reading frame abolished
TRP4
mRNA 3
'
-end formation in forward orientation and had no effect on the reverse orientation.
INTRODUCTION
Yeast messenger RNA 3'-end formation resembles the process in mammals in many aspects. It
has been shown for several yeast mRNAs that mRNA 3'-end formation requires cleavage of a precursor RNA and
polyadenylation. There are, however, some remarkable differences between mRNA 3'-end formation in mammals and in yeast. Whereas cleavage and
polyadenylation are strongly coupled in mammals (
1
), they can be uncoupled in yeast (
2
). No universal sequence motif exists that corresponds to the AATAAA
polyadenylation signal in higher eukaryotes but various different sequence
motifs involved in mRNA 3'-end formation have been identified in the yeast
Saccharomyces cerevisiae
. These motifs TAGTA, TATATA, TACATA, TATGTA, TACGTA (
3
-
5
), TTTATA, TATGTT, TATTTA (
6
), TTTTTATA (
7
) and (AT)
9
(
8
,
9
) show a high content in A and T nucleotides. In initial studies by Zaret and
Sherman (
5
) a tripartite sequence motif TAT...TA(T)GT...TTT has been proposed to be
involved in 3'-end formation. In the cauliflower mosaic virus (CaMV) a condensed
version of the tripartite motif has been shown to be essential and we have
shown that an exchange from a T to a C nucleotide within such motifs reduces 3'-end formation activity (
10
). In different studies it has been shown that several of these sequence
elements act in concert to result in full activity (
6
,
10
,
11
). Signal sequences for mRNA 3'-end formation are degenerate and present in several copies at the 3'-ends of RNA polymerase II transcribed genes in yeast.
In contrast to the highly conserved mammalian hexanucleotide sequence motif
where single point mutations mostly abolish function, only one single point
mutation was reported to dramatically reduce 3'-end formation in an authentic gene of the yeast
S.cerevisiae
. This point mutation was found in the
ADH2
3'-untranslated region (3'-UTR) (
12
).
We have shown that 3'-end formation elements in yeast can be grouped into two
functionally different classes (
13
). The 3'-end formation elements of one class direct 3' processing only in one orientation (unidirectional) in an
in vivo
test system, whereas 3'-end formation elements of the other class act in both orientations (bidirectional). A representative of the class of unidirectional 3'-end formation elements is the
GCN4
site. This element is highly complex and contains two copies of the signal
sequence TTTTTAT interrupted by a region which is responsible for poly(A) site
selection (
14
,
10
). The 3'-end regions of genes from the class of bidirectional 3'-end formation sites,
TRP1
,
TRP4
and
ARO4
, all contain at least one TA(T)GT sequence motif on their sense as well as on
their antisense DNA strand. We have proposed that these motifs might be a
prerequisite for the bidirectional function of these elements (
13
). This prompted us to analyse the effects of mutations within these elements.
In this report, we have analyzed and defined the bidirectional
TRP4
3'-end formation element in both orientations in more detail. We were interested how mutations within this region would affect 3'-end formation function in both orientations. We
hypothesized that mutations resulting in different effects on polyadenylation
for each orientation would suggest the existence of two distinct 3'-end formation signals for each orientation. Similar effects of
mutations on both orientations support the existence of a single 3'-end formation signal.
We found that the
TRP4
3'-end formation element can be subdivided into two different regions.
The essential sequence motifs of both regions are TAGT sequence motifs. One
region is located between the translational stop codon and the major poly(A)
site. The poly(A) site itself is not required for function. Most changes within
this region affect mRNA 3'-end formation in both orientations. Changes in the other region
affect mRNA 3'-end formation only in the forward orientation. Surprisingly, this
region is located at the end of the
TRP4
open reading frame (ORF) which is a novel feature of yeast 3'-end formation sites.
MATERIALS AND METHODS
Yeast strains, media and methods
The yeast strain RH1376 has been previously described (
10
) and is a derivative of the
S.cerevisiae
laboratory standard strain X2180-1A (
MAT
a
gal2
SUC2
mal
CUP1
). Yeast strains were cultivated in YEPD complete medium or MV minimal medium (
15
). Yeast transformation (
16
), DNA isolation (
17
) and Southern analysis (
18
) were previously described.
Enzymes and oligonucleotides
Restriction enzymes were purchased from Boehringer (Mannheim, Germany) and New
England BioLabs (Schwalbach, Germany). Vent DNA polymerase was purchased from
New England BioLabs. Oligonucleotides were synthesized by Microsynth (Balgach, Switzerland).
Plasmid construction and cloning
A 270 base pair (bp) fragment from the
Bst
EII to the
Eco
RV of the
TRP4
3'-end region was amplified using the polymerase chain reaction (
19
) and the two oligonucleotide primers NAT1 and NAT2 containing
Xho
I restriction sites at their ends. This fragment was cleaved with
Xho
I and inserted in either orientation into the
Xho
I digested plasmid pME729 (
10
). The resulting vector was the basis for the construction of the various
TRP4
point mutations in this work. All point mutations, small deletions and
insertions were created using oligonucleotide primers that contained the
desired mutations by the polymerase chain reaction according to Giebel and
Spritz (
20
). The 5' and 3' deletions were constructed as follows. The
TRP4
element was amplified from position -111 to +191 using appropriate oligonucleotides, restricted with
Xho
I and
Eco
RV, and cloned into the
Xho
I/
Sma
I sites of the vector pGEM7
®
-7Zf(+) (Promega, Madison, WI, USA). The plasmid was linearized with
Bam
HI for the 5' deletions or with
Xba
I for the 3'-end deletions and treated with the
Bal
31 exonuclease enzyme. The ends of the shortened plasmids were made blunt and
ligated with a
Pst
I linker [5'-d(GCTGCAGC)-3'] for the 5' deletions and with a
Pst
I and
Hin
dIII linker [5'-d(CCAAGCTTGG)-3'] for the 3' deletions. The screened deletion fragments
were isolated using the restriction enzymes
Pst
I and
Hin
dIII and cloned in both orientations into the test construct on the plasmid
pME729 which was cut with the enzymes
Pst
I and
Hin
dIII. All constructs were verified by DNA sequencing (
21
).
RNA analysis
Total RNA from
S.cerevisiae
was isolated according to Furter
et al.
(
22
). For Northern hybridization experiments, ~10 [mu]g total RNA was precipitated, resuspended and denatured in 30 [mu]l sample buffer [50% v/v deionized formamide, 6% v/v formaldehyde, 1* loading buffer, 10% (v/v) TE buffer] for 5 min at 65oC and put on ice. The RNA was separated on a denaturing
formaldehyde agarose gel. The 1.4% w/v agarose gel [3% (v/v) formaldehyde, 20
mM MOPS, 5 mM Na-acetate, 1 mM EDTA) was run for 3 h at 60 V in a buffer containing 20 mM
MOPS, 5 mM Na-acetate and 1 mM EDTA. The gel was soaked twice in 25 mM Na-phosphate buffer for 20 min each time and the RNA was transferred
onto a nylon membrane (Amersham, UK) by electroblotting (2 A, 50 V) for 3-4 h in 25 mM Na-phosphate buffer. After washing in 2* SSC, drying on 3MM paper and crosslinking under UV light
(254 nm) for 5 min, the membrane with the bound RNA was hybridized at 42oC with a labeled fragment for 24 h in 50 ml hybridization mix [50% v/v
formamide, 50 mM Na-phosphate pH 6.5, 800 mM NaCl, 1 mM EDTA, 0.5% (w/v) SDS, 10* Denhardt's solution, 150 [mu]g/ml calf thymus DNA, 500 [mu]g/ml torula yeast RNA]. The 440 bp
Mlu
I-
Xho
I DNA fragment of the
ACT1
5' region was randomly radiolabeled as described (
23
). The RNA was visualized by autoradiography. Band intensities from
autoradiographs were quantified using a phosphoimager (Molecular Dynamics, Sunnyvale, CA, USA).
RESULTS
The
TRP4
3'-end formation element causes 3'-end formation in an orientation-independent manner in an
in vivo
test system (
13
). An additional puzzling characteristic of this 3'-end formation site is that no
in vitro
processing could be detected (data not shown) using various yeast cell extracts
which were able to process transcripts containing other polyadenylation sites
derived from
GCN4
,
CYC1
or
ADH1
(
14
).
This prompted us to ask (i) what mutations within this element would affect 3'-end formation function in both orientations, and (ii) what sequence
requirements are responsible for this bidirectional function? The
TRP4
gene 3'-UTR contains three TA(T)GT(T/A)(T) sequences, originally proposed
by Zaret and Sherman (
5
) to be involved in 3'-end formation. One motif is located 12 bp downstream of the
translational stop codon (
13
). The other two motifs are located in inverse orientation 24 and 49 bp
downstream of the A of the translational stop codon (Fig.
1
). The most upstream motif TATGTT was named ZS1, the proximal inverse TAGTTT
motif was referred to as ZS2, and the distal TAGTA motif ZS3. In addition, a
TAGT motif is located in the
TRP4
ORF starting at position -17.
DISCUSSION
The sequence requirements for yeast mRNA 3'-end formation sites have been studied for a long period of time.
The analysis has been difficult because many yeast elements seem to be complex
consisting of numerous redundant elements. For the 3'-end formation site of the yeast
GCN4
gene encoding a transcriptional regulator we have recently shown that ~100 nt are required for fully active 3'-end formation function (
14
). An additional difficulty in the analysis of yeast polyadenylation sites is the variety of degenerate
sequence motifs and their different functions. Several lines of evidence
suggest at least three different roles for sequence motifs in 3'-end formation. One has to differentiate between efficiency elements
of 3'-end formation with various TA-rich sequence motifs (
3
,
5
,
11
,
14
), elements of various A-rich sequences which select the poly(A) addition site (
14
,
24
), and the actual poly(A) site itself which seems to prefer PyA sequences (
25
).
In this study we have focused on the capacity of the
TRP4
element to direct 3'-end formation in both orientations which is a special feature of
numerous yeast polyadenylation sites. Our aim was to find out what sequence
determinants are required for a yeast 3'-end formation site to be able to function in both orientations.
With our experiments we could not discriminate between the various processes in
mRNA 3'-end formation including processing, polyadenylation and termination
of the precursor transcript. We wanted to know whether there is a common feature for both orientations in such 3'-end formation sites or whether we have simply two independent and
overlapping sites. Bidirectional function is a characteristic for numerous but
not for all yeast 3'-end formation sites. We have analyzed the
TRP4
3'-end formation element as an example for such a symmetrical element. Other bidirectional 3'-end formation elements are the Ty retroelement (
26
) and the
ARO4, TRP1,
CYC1
,
GAL1
,
GAL7
and
GAL10
polyadenylation elements (
13
,
27
). An example of a 3'-end formation signal of the other class is the complex
GCN4
polyadenylation site (
14
) which functions very efficiently but exclusively in the natural forward
orientation. The significance of bidirectional signals for mRNA 3'-end formation in yeast is still unclear. No convergent transcripts
have been identified downstream of the
TRP4
and of the
GAL
genes (
13
,
27
). Bidirectional 3'-end formation signals might have a safety function in the cell,
namely to prevent that any RNA polymerases transcribe from the 3'-end into a gene and interfere with transcription of this gene.
Our results have shown that the bidirectional
TRP4
3'-end formation element consists of two different parts. The region
of ~70 nt located in the 3'-UTR between the translational stop codon of the
TRP4
ORF and the major poly(A) addition site is required for 3'-end formation in both orientations. The site of poly(A) addition is
not necessary for efficient 3'-end formation in either orientation. In this region a TAGT(T) motif
(ZS2) in inverse orientation is flanked by a proximal TATGT motif (ZS1) and a
distal TAGTA motif (ZS3) (Fig.
1
). The central TAGTT motif is essential for 3'-end formation in both orientations and does not tolerate point
mutations without drastic loss of function in both orientations. A single point
mutation destroying 3'-end formation function in forward orientation was observed up to
now in only one yeast gene i.e. in the
ADH2
3'-end formation element (
12
). For the
GCN4
3'-end formation element which we have analysed recently (
14
) we could not find such drastic effects on 3'-end formation function. This suggests that the unidirectional
GCN4
element seems to include more redundant signals for interaction with the 3'-end formation machinery than the
TRP4
element. One possibility to explain the result that the point mutations within
the ZS motifs of the
TRP4
element as well as 5' and 3' deletions of this region had significant effects in both
orientations is the existence of a higher order RNA structure defined by the
TRP4
element. The ZS2 TAGT motif is the essential element of this region. It might
be located at an exposed position and seems to be a good candidate for an
interaction site between RNA and the proteins of the 3'-end formation machinery. The ZS1 and ZS3 motifs seem to contribute
as auxiliary elements. Small changes in the spacing between ZS1 and ZS2 affect
function in forward and reverse orientations differently. Whereas 3'-end formation function in forward orientation is significantly decreased, the function in
the reverse orientation is even improved. We assume that differences in spacing
destroy a higher order RNA structure of this region required for the
bidirectional function.
Additional sequences located in the 3' terminal part of the
TRP4
ORF are required for 3'-end formation in forward orientation. ORF sequences have not been
reported so far to be involved in 3'-end formation
in vivo
. For the
GAL1
and
GAL10
3'-end formation elements 3' processing was reported to be reduced in an
in vitro
reaction when ORF sequences were deleted (
27
). Presumably this effect was dependent on differences in the transcript length
of various transcripts tested. In the case of the
TRP4
ORF element transcript length has not been changed. The effect of a loss of
polyadenylation function specifically in forward but not in reverse orientation
was caused by a single point mutation. Interestingly this point mutation was
also located in the ZS core motif TAGT. Exchanges in all other TA-sequences in the 3' terminal part of the ORF did not have any significant effect on 3'-end formation.
Both TAGT motifs in the ORF as well as ZS2 in the untranslated region are
essential for the function in forward orientation. In reverse orientation 3'-end formation seems to depend only on one motif, the ZS2 TAGT motif
in the UTR. This might be one reason why the efficiency of mRNA 3'-end formation in the test system of the wild-type element in the reverse direction is lower when compared
to the forward orientation.
ACKNOWLEDGEMENTS
We thank Christoph Springer and Chris Berens for critical reading of the
manuscript. This work was supported by the Deutsche Forschungsgemeinschaft and
by the Fonds der Chemischen Industrie.
REFERENCES
1 Wahle,E. and Keller,W. (1992) Annu. Rev. Biochem.61, 419-440.MEDLINE Abstract
2 Egli,C.M. and Braus,G.H. (1994) J. Biol. Chem.269, 27378-27383.MEDLINE Abstract
3 Irniger,S. and Braus,G.H. (1994) Proc. Natl. Acad. Sci. USA91, 257-261.MEDLINE Abstract