From the Department of Molecular and Cellular
Biochemistry, The Comprehensive Cancer Center, and the
§ Molecular, Cellular, and Developmental Biology Graduate
Program, The Ohio State University, Columbus, Ohio 43210
Received for publication, November 20, 2000
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ABSTRACT |
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Previous work from this laboratory identified a
polysome-associated endonuclease whose activation by estrogen
correlates with the coordinate destabilization of serum protein
mRNAs. This enzyme, named polysomal ribonuclease 1, or PMR-1, is a
novel member of the peroxidase gene family. A characteristic feature of
PMR-1 is its ability to generate in vitro degradation
intermediates by cleaving within overlapping APyrUGA elements in the
5'-coding region of albumin mRNA. The current study sought to
determine whether the in vivo destabilization of albumin
mRNA following estrogen administration involves the generation of
decay intermediates that could be identified as products of PMR-1
cleavage. A sensitive ligation-mediated polymerase chain reaction
technique was developed to identify labile decay intermediates, and its
validity in identifying PMR-1-generated decay intermediates of albumin
mRNA was confirmed by primer extension experiments performed with
liver RNA that was isolated from estrogen-treated frogs or digested
in vitro with the purified endonuclease. Ligation-mediated
polymerase chain reaction was also used to identify decay intermediates
from the 3'-end of albumin mRNA, and as a final proof of principle
it was employed to identify in vivo decay intermediates of
the c-myc coding region instability determinant
corresponding to sites of in vitro cleavage by a
polysome-associated endonuclease.
The process of mRNA decay is closely linked to translation
(1), and a growing body of data links the binding of proteins to the
mRNA 3'-end with the efficiency of cap-dependent
translation initiation (2, 3). mRNA decay in vertebrates can be
generally divided into four basic pathways: deadenylation with the
possible link to decapping and 5'-3' degradation of the mRNA body,
deadenylation followed by 3'-5' decay of the mRNA body,
nonsense-mediated decay, which may or may not involve decapping, and
endonuclease-mediated decay (4-6). All of these effectively serve to
disrupt the link between the 5'- and 3'-ends of the mRNA. It has
yet to be proven whether the general pathway of mRNA decay in
vertebrates will recapitulate processes that have been characterized in
yeast, but there is suggestive evidence to support this.
A significant number of mRNAs are also degraded through
endonuclease-mediated pathways (5). Examples include mRNAs for apo-very low density lipoprotein II (7), c-myc (8),
transferrin receptor (9), insulin-like growth factor II (10), and
With the exception of insulin-like growth factor II mRNA, whose
unique structure results in a remarkably stable in vivo
endonuclease degradation product (10, 16), most mRNAs are degraded
without significant accumulation of decay intermediates. However, decay intermediates have been observed using crude in vitro decay
systems. The most likely explanation for this is that in
vivo these intermediates are subject to rapid exonucleolytic
clearance in a manner similar to that which occurs in prokaryotes,
where polynucleotide phosphorylase or RNase II rapidly degrade
intermediates generated by RNase E (5). For mRNAs that do not show
obvious degradation intermediates only a few, highly abundant mRNAs
have been examined, in general using primer extension or S1 nuclease
protection assays (17). Although these techniques do work, they require
long exposure times to visualize metastable products and are not
readily applicable to low copy mRNAs. We describe here a new
approach using ligation-mediated (LM) RT-PCR to identify the 3'-ends of
degradation intermediates generated in vivo as a result of
either endonuclease cleavage or pausing of a 3'- to
5'-exonuclease. The rationale behind this was our finding that
PMR-1 generates degradation intermediates with 3'-hydroxyls, which are
good substrates for further degradation by 3'- to 5'-exonucleases,
whereas decay intermediates with 3'-phosphate termini are poor
substrates for such enzymes. Using this assay we show that the
estrogen-induced increase in unit activity of polysome-bound PMR-1 is
accompanied by the appearance of albumin mRNA degradation
intermediates corresponding to those generated by in vitro
cleavage with the purified endonuclease. As a proof of principle this
approach was used to demonstrate in vivo cleavage of
c-myc mRNA in the coding region determinant that has
been identified from in vitro studies as a potential
regulatory element by Ross and coworkers (8, 18).
Experimental Animals--
Male Xenopus were obtained
from Xenopus One (Ann Arbor, MI), fed a synthetic diet, and maintained
in plastic aquaria with a 12-h light-dark cycle. One milligram of
estradiol-17 Purification of Xenopus Liver RNA--
Livers were removed and
perfused with ice-cold 1× SSC to remove as much blood as possible.
They were chopped into 1-mm cubes followed by the addition of 10 ml/g
of tissue of 5 M guanidine isothiocyanate, 50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 5 mM Cell Culture--
Murine erythroleukemia cells (MEL cells) were
obtained from Dr. Lynne Maquat. They were cultured in Dulbecco's
minimal essential medium containing 10% fetal bovine serum. Total
cellular RNA was isolated using TRIzol reagent (Life Technologies,
Inc.) following the manufacturer's protocol.
Primer Ligation--
Between 2 and 10 µg of total RNA was
added to a 15-µl reaction containing 50 mM Tris, pH 8, 10 mM MgCl2, 20 mM ATP, 2 mM DTT, 10 µg/ml BSA, 1 mM hexamine cobalt
chloride, 25% (w/v) polyethylene glycol 8000, 30 units of placental
ribonuclease inhibitor, and 2 µg of ligation primer MH11NH3P. The
ligation primer MH11NH3P (P-CCAGGTGGATAGTGCTCAATCTCTAGATCG-NH3)
was prepared by Operon and has 5'-phosphate and 3'-amino termini.
Ligations were performed at 4 °C for 16 h with 15 units of T4
RNA ligase (Life Technologies, Inc.). The reaction mixture was
extracted once with one volume of phenol:chloroform:isoamyl alcohol
(25:24:1) and once with one volume of chloroform:isoamyl alcohol
(24:1). The aqueous layer was adjusted to 0.3 M sodium
acetate, pH 5.5, and RNA with the ligated primer was precipitated by
addition of 2.5 volumes of ice-cold ethanol.
RT-PCR--
The entire mixture of RNA ligated to the MH11NH3P
primer was resuspended in 11 µl of DEPC-treated water, followed by
addition of 250 ng of the primer MH12 (5'-CGAGCTAGAGATTGAGCAC), which
is complementary to the first 19 nt of MH11NH3P. The solution was heated to 100 °C for 3 min and quickly cooled on ice. To this was
added 4 µl of 5× buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2, 10 mM DTT), 0.5 mM dNTPs, and H2O to a
total volume of 20 µl. The mixture was heated at 42 °C for 2 min
followed by the addition of 200 units of Superscript II reverse
transcriptase (Life Technologies, Inc.) and incubated for an additional
50 min. The reaction was terminated by heating at 70 °C for 15 min.
Three µl of the above reaction mixture was mixed with 2.5 µl of
10× buffer (100 mM Tris-HCl, pH 8.9, 1 M KCl,
15 mM MgCl2, 500 µg/ml BSA, 0.5% Tween 20 (v/v)). To this was added MgCl2 and dNTPs to a final
concentration each of 3 mM. The wax bead of the Hot Start tube (Life Technologies, Inc.) was melted by heating the tube to
75 °C for about 30 s, and the mixture was cooled quickly on ice. 11 µl of DEPC water, 2 µl of 5'-32P-labeled
gene-specific primer, and 0.5 µl of tTh polymerase from Roche
Molecular Biochemicals (Indianapolis, IN) were then added to a final
volume of 25 µl. The mixture was heated at 95 °C for 2 min, and
PCR amplification was performed for 25 cycles at 95 °C for 2 min,
58 °C for 30 s, and 72 °C for 1 min, followed by extension
for 3 min at 72 °C using primers synthesized by Operon Technologies
(Alameda, CA). The gene-specific primers used were, Set A1
(5'-CGCGGTACCTGGATCACCCTGATTTGTC, beginning at position 40 of albumin
mRNA), Set G1 (5'-TCCTTGTGAAGCTGATTA, beginning at position 1690 of
albumin mRNA), and MH28 (5'-AAGAGGCGAACACACAACG, beginning at
position 1669 of c-myc mRNA). The reaction mixtures were
then extracted as above with phenol:chloroform:isoamyl alcohol, and
amplified products were recovered by ethanol precipitation. The
recovered pellet was dissolved in 3 µl of DEPC H2O, and
an equal volume of formamide loading dye (80% formamide, 1 mM EDTA, pH 8, 0.1% bromphenol blue, and 0.1% xylene
cyanol). This was heated for 5 min at 95 °C and loaded into a single
lane of a denaturing 6% polyacrylamide/urea gel.
Product Recovery, Reamplification, and Sequencing--
Products
from the above reaction were identified by autoradiography of the dried
polyacrylamide gel. The bands of interest were extracted from the dried
gel following a modified protocol from Jo et. al. (19). The
film was aligned with the gel, and an 18-gauge needle was used to punch
holes at the four corners of each band. A fresh razor blade was then
used to cut out each desired band, and the excised gel fragments were
soaked in 200 µl of distilled H2O for 10 min. The tube
was then boiled for 15 min with Parafilm to hold the lid closed.
This was then centrifuged at 10,000 × g for 2 min, and
the supernatant was removed. DNA was recovered by addition of one-tenth
volume of 3 M sodium acetate, pH 5.5, 50 µg of glycogen,
and 900 µl of 100% ethanol, followed by chilling at
Four microliters of the extracted DNA was added to a 40-µl reaction
containing 4 µl of a 10× buffer consisting of 100 mM
Tris-HCl, pH 8.9, 1 M KCl, 15 mM
MgCl2, 500 µg/ml BSA, 0.5% Tween 20 (v/v). The reaction
mixture was adjusted to 1.5 mM MgCl2, followed
by the addition of 0.4 mM dNTPs, 1 ng of primer MH12, 1 ng
of the gene-specific primer, and 2.5 units of tTh polymerase (Roche
Molecular Biochemicals, Inc.). PCR amplification was performed as
described above, following which 8 µl was treated with 10 units of
exonuclease I and 2 units of shrimp alkaline phosphatase for 20 min at
37 °C to remove unincorporated primers. This reaction was terminated by heating for 15 min at 80 °C, followed by addition of 20 ng of the
original gene-specific primer. This was annealed to the template, by
heating at 100 °C for 3 min and then cooling on ice for 5 min, and
sequenced using T7 DNA polymerase (USB T7 Sequenase Kit). The products
were denatured and electrophoresed on a 6% polyacrylamide/urea gel as
described above.
In Vitro Cleavage of the 3'-End of Albumin mRNA--
A
5'-32P-labeled transcript corresponding to region G in Fig.
2 (spanning 1690-2002 of albumin mRNA) was prepared as described previously (17). This was incubated with 10 µg of polysome extract from estrogen-treated frogs (Fig. 5A) or 20 units of PMR-1
purified as described previously (20, 21) (Fig. 5B) in 30 mM Tris-HCl, pH 7.5, 1 mM DTT, 2 mM
MgCl2, and 75 mM KCl, at 23 °C. One unit of
PMR-1 is the amount needed to completely cleave 7 fmol of albumin substrate transcript in 30 min at 23 °C. The reactions were stopped by adding one volume of stop solution (98% formamide (v/v), 0.1% (v/v) bromphenol blue, and 0.1% xylene cyanole) and heating at 95 °C for 3 min. The samples were then electrophoresed on a 6% polyacrylamide-urea gel, and cleavage products were visualized by autoradiography.
RNase T1 Digestion and RNA Structure Modeling--
A
5'-32P-labeled transcript corresponding to nt 1690-2002 of
albumin mRNA was added to 10 µl of hybridization III buffer from the Ambion RNase Protection Assay III kit (Ambion, Austin, TX). This
was heated for 5 min at 50 °C and cooled slowly to room temperature. To this was added 150 µl of RNase digestion buffer III containing 5 units of RNase T1. Samples were incubated for 5-25 min at 37 °C,
and the reaction was stopped by addition of 225 µl of RNase inactivation/precipitation III solution. Digested RNA was recovered by
addition of 150 µl of ethanol, and 10 µg of yeast tRNA, followed by
precipitation at Primer Extension--
Primer extension analysis of albumin
mRNA was performed as described previously (17). 10-µg samples of
total liver RNA isolated 12 h after injection of estradiol were
either kept on ice or digested as described above with 20 or 40 units
of purified PMR-1. The mixtures were heated to inactivate PMR-1, and
ethanol was precipitated with 1-2 × 105 dpm of
5'-32P-labeled primer DAN25 (5'-CACTCAGGAGTTTTGTCATTAA),
which is complementary to nucleotides 280-301 of albumin mRNA. The
precipitated RNA and primer were dissolved in 10 µl of annealing
buffer (50 mM Tris-HCl, pH 8.7, 0.54 M KCl, 1 mM EDTA), heated at 65 °C for 10 min, and slowly cooled
to 25 °C. To each tube was added a mixture of 0.9 mM
dATP, dCTP, dGTP, TTP/50 mM Tris-HCl, pH 8.3/13
mM MgCl2/7 mM dithiothreitol and
200 units of Moloney murine leukemia virus reverse transcriptase to a
total volume of 40 µl. The reaction mixture was incubated for
1.5 h at 42 °C and stopped by the addition of 260 µl of stop
solution (0.3 M sodium acetate, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Products were recovered by
precipitation with 600 µl of cold ethanol. The final pellets were
dissolved in 6 µl of formamide loading buffer and electrophoresed on
a 6% acrylamide/urea gel. The position of primer extension stops and
nuclease cleavage sites was determined relative to a sequencing ladder
prepared from the cloned cDNA and the same primer.
A Sensitive LM-PCR Assay for Detecting in Vivo mRNA Degradation
Intermediates--
Earlier work found that degradation intermediates
generated by in vitro PMR-1 cleavage of albumin mRNA
contained free 3'-hydroxyls, making them susceptible to degradation by
3'- to 5'-exonucleases (23). We took advantage of this observation to
develop a generally applicable approach to the identification of
in vivo mRNA degradation intermediates. The method
diagrammed in Fig. 1 involves ligation of
a common primer to the 3'-ends of all RNA molecules followed by reverse
transcription primed with a nested complementary primer to generate a
population of cDNAs corresponding to all of the primer-tagged RNA
fragments in the population. The cDNA is PCR-amplified using a
5'-32P-labeled sense-strand primer specific to the mRNA
of interest and the nested primer used for reverse transcription, and
the resulting products are separated on a denaturing
polyacrylamide/urea gel. Bands identified by autoradiography are
excised, re-amplified, and sequenced to identify the 3'-end of the
mRNA degradation intermediate at the junction between the ligated
primer and the target mRNA. Key to this process is the use of a
ligation primer that has been modified by addition of a 5'-phosphate
for the ligation to the 3'-hydroxyl termini, and a 3'-amino group to
prevent further multimerization of the primer during ligation. This
technique does not distinguish between decay intermediates generated by
endonuclease cleavage versus those produced by pausing of a
3'- to 5'-exonuclease, but as noted under "Discussion," the latter
have not been detected in mRNAs bearing poly(G) tracts.
LM-PCR Identification of in Vivo Albumin mRNA Decay
Intermediates--
PMR-1 was identified and purified based on its
ability to generate a unique product by cleaving within overlapping
APyrUGA elements in the 5'-coding region of the molecule (14, 20). Although PMR-1 cleaves preferentially within APyrUGA elements, it is
not a restriction endonuclease and also cleaves at numerous "nonconsensus" elements (15, 23). Fig.
2 shows a schematic representation of
albumin mRNA with the positions of the 14 APyrUGA elements
indicated in relation to the coding (gray) and noncoding (black) regions. All of these elements fall within the
coding region, and there are three pairs of overlapping elements
(identified with brackets). Because the in vitro
cleavage properties of PMR-1 were characterized with the portion of the
5'-coding region, identified as A in Fig. 2, this sequence
was evaluated for the appearance of in vivo degradation
intermediates following estrogen administration. Male
Xenopus were injected with 1 mg of estradiol, and total
liver RNA isolated 12 or 24 h later was analyzed by LM-PCR using
the A1 primer (indicated with a filled horizontal arrow in
Fig. 2) beginning at position 40 of albumin mRNA. Lung RNA isolated
at time 0 was used as a control. Fig.
3A shows the gel separation of
RT-PCR products generated by amplification with 32P-labeled
A1 primer. The bands numbered on the right side of the autoradiogram
correspond to the cleavage sites mapped in Fig. 3C onto
structure of the corresponding region of albumin mRNA. A
nonspecific product distinct from albumin mRNA was seen to varying degrees in all samples (filled circle). Two major
amplification products from time 0 liver RNA were seen in the
lower third of the gel, one of which corresponded to
position A14, and the other of which was too close to the sequencing
primer to be identified (open circle). Of note was the
time-dependent appearance of new products 12 and 24 h
after estrogen administration (lanes 3 and 4).
Each of the indicated bands was excised from the gel, amplified, and
sequenced to identify the junction between the ligated primer and
albumin mRNA degradation products. An example of this is shown in
Fig. 3B for band A11. The sequence
5'-AUU-primer-3', corresponded to cleavage 5'-AUU/GAACUGA-3'
within the first APyrUGA element in the stem-loop of this portion of
albumin mRNA. The positions of the in vivo cleavage
sites identified in region A are indicated in Fig. 3C with
numbered arrows corresponding to the LM-PCR products in Fig.
3A. The filled arrows on this figure identify
in vivo sites that correspond to the main sites of in vitro cleavage by purified PMR-1.
Previous work characterized PMR-1 as cleaving between the two
pyrimidines in the APyrUGA consensus element (23). The absolute identification of cleavage sites afforded by the sequencing of LM-PCR
products indicated that this was misplaced by a single nucleotide, a
result that likely came from the use of DNA markers and sequencing
ladders to size RNA degradation intermediates. The actual cleavage lies
between the U and G residues, a result that is consistent with
inactivation of cleavage upon mutating APyrUGA to APyrAGA (23). The
corrected cleavage sites are indicated in all the figures presented here.
Primer Extension Confirms the LM-PCR Identification of in Vivo
PMR-1 Cleavage Sites--
In the experiments in Fig.
4 primer extension was used to both
validate the LM-PCR identification of in vivo decay
intermediates and to show that these resulted from cleavage by PMR-1.
In the experiment in Fig. 4A total liver RNA was isolated
from control frogs or frogs injected with estradiol 12 h prior to
death, and primer extension was performed using a primer for region A
indicated by the open arrow above the schematic for albumin
mRNA in Fig. 2. The positions of the cleavage site were determined
relative to a DNA sequencing ladder prepared from albumin cDNA
using the same primer. Although the 12-h post-estrogen liver RNA had
little intact full-length albumin mRNA, both this and control RNA
display equal signal intensities for products that extend from the
primer to the cap site. Because the primer extension will detect all mRNA decay intermediates upstream from the 5'-end of the primer at
position 301, these results indicate that there is a substantial population of 5' decay intermediates that are not detected as discrete
products by Northern blot. In agreement with the results in Fig. 3,
there was little evidence for decay intermediates within the 5' 300 nt
of albumin mRNA extracted from control animals, and RNA from 12-h
estrogen-treated frogs displayed the same in vivo decay
intermediates by primer extension (numbered on the side) as
those identified in Fig. 3 by LM-PCR.
To determine whether these products resulted from in vivo
cleavage by PMR-1 we subjected the 12-h post-estrogen liver RNA to
in vitro digestion with purified PMR-1. Because this
preparation retained a significant amount of the first 300 nt of
albumin mRNA, we reasoned that further in vitro
digestion with purified PMR-1 should amplify the signal intensity of
products generated by PMR-1 cleavage in vivo. Alternatively,
if these decay intermediates were not generated by PMR-1, further
in vitro digestion with the purified enzyme would generate a
different set of primer extension products. The results of digestion
with 40 units of purified PMR-1 are shown in lanes 1 and
2 of Fig. 4B. In vitro cleavage of RNA that was partially "precleaved" in vivo resulted in
increased signal intensity for all of the products identified by both
LM-PCR and primer extension. Because previous work showed that these decay products are unique to PMR-1 (23) these results indicate that the
identified decay intermediates resulted from in vivo cleavage by PMR-1.
Application of LM-PCR to Identify Degradation Intermediates from
the 3'-End of Albumin mRNA--
Unlike region A in the 5'-coding
portion of albumin mRNA, metastable degradation intermediates were
never observed in the course of in vitro decay experiments
using transcripts from the 3'-end of albumin mRNA (14). Region G
(Fig. 2), spanning position 1690 to 2002 of albumin mRNA, contains
173 nt of the coding region and the 139-nt 3'-UTR, and has four APyrUGA
elements. In vivo cleavage within this sequence was analyzed
by LM-PCR in Fig. 5 using the RNA from
24-h estrogen-treated frogs evaluated in Fig. 3. It should be noted
that the 6% polyacrylamide gels used here and in Fig.
6 are only capable of resolving cleavage
at three of the four APyrUGA sites. Eight LM-PCR products were
identified in the gel in Fig. 5A, and their positions in the
sequence of region G are indicated in Fig. 5B. Site G1
corresponds to the 3'-end of albumin mRNA, and sequencing of this
confirmed the presence of the <17-nt poly(A) tail seen in our previous
work (24). Like the consensus sites A10 and A11 in the 5'-coding
region, there are two overlapping APyrUGA elements in this portion of
albumin mRNA (sites G2 and G3). However, neither of the LM-PCR
products corresponding to cleavage at G2 and G3, nor the product for
cleavage at the other APyrUGA element G7, were as prominent as that
seen for A11 in Fig. 3A.
In Vitro Cleavage of the 3'-End of Albumin mRNA--
To
determine why results with region G did not match those seen with
region A, we determined the pattern of in vitro cleavage of
region G of albumin mRNA using 5'-end-labeled transcript in a
manner similar to that used earlier to study cleavage within region A
(23). In the experiment in Fig. 6A, region G transcript was
incubated with polysome extract from estrogen-treated frogs prepared as
described previously (14). Degradation intermediates corresponding to
the in vivo cleavage sites identified in Fig. 5 are labeled
on the right. Although numerous bands were seen that are
typical for in vitro assays, the main pattern was similar to
that observed for in vivo decay by LM-PCR. This experiment was repeated in Fig. 6B using 20 units of purified PMR-1
instead of polysome extract. Here only two major cleavages were
observed, one at site G7, which contains a consensus APyrUGA element,
and one at the adjacent site G8. Based on results obtained in Figs. 3
and 4, we suspect that 1) the better correspondence between the
cleavage pattern seen with polysome extract and in vivo
decay intermediates either resulted from the presence of auxiliary
proteins in the extract that potentiate the ability of PMR-1 to cleave this portion of albumin mRNA or 2) some of these bands are products of additional steps in mRNA decay.
Impact of Secondary Structure on PMR-1 Cleavage of the Albumin
mRNA 3'-End--
When APyrUGA is present in a single-stranded
conformation, it is the predominant site for PMR-1 cleavage in both
albumin (14) and vitellogenin mRNA (25); however, PMR-1 is unable
to cleave within the element when present in double-stranded RNA (23). To test whether the lack of strong cleavages within APyrUGA sites in
region G was due to RNA secondary structure, a 5'-end-labeled transcript was digested with limiting amounts of RNase T1, and the
identified single-stranded G residues were used to model this sequence
to secondary structure with the MFOLD program (22). The results of the
T1 digestion are shown in Fig.
7A, and the derived secondary
structure is shown in Fig. 7B. In this structure the sites
cleaved by RNase T1 sites are identified as H, the APyrUGA elements are boxed, and the in vivo cleavage
sites are indicated with arrows. Both the APyrUGA element at
G7 and the adjacent nonconsensus site G8 are in a large single-stranded
bulge. The overlapping APyrUGA sites at G2 and G3 are in a constrained
hairpin structure that would prevent cleavage by PMR-1 in a manner
similar to that observed previously (23). This accounts for the minimal
cleavage observed here in Fig. 6. Although the fourth consensus site
was present in this transcript, it was not resolved in Fig. 6; this element too was structurally constrained and unlikely to be cleaved in vitro by PMR-1. Taken together, these results indicate
structural constraints within the 3'-end of albumin mRNA impact on
its ability to be cleaved by PMR-1. Nevertheless, the in
vitro decay pattern matches that observed in vivo by
LM-PCR.
Identification of Decay Intermediates Consistent with in Vivo
Endonuclease Cleavage within the c-myc CRD--
Ross and coworkers
identified an mRNA instability determinant within the coding region
of c-myc mRNA (the coding region determinant (26)) that
is both a site for in vitro cleavage by a
polysome-associated endonuclease activity (8) and for binding by a
KH-domain protein (18). Because little was known about the relationship
between the CRD and the degradation of c-myc in vivo, we
chose to examine cleavage within the CRD as a test of the ability of
LM-PCR to detect labile in vivo decay intermediates from a
rare and inherently unstable mRNA. In the experiment in Fig.
8 LM-PCR was performed on 2 µg of total
RNA isolated from murine erythroleukemia (MEL) cells using a
gene-specific primer complementary to nucleotides 1669-1687 of
c-myc mRNA. A denaturing polyacrylamide gel of the 32P-labeled LM-PCR products is shown in Fig. 8A,
and the sequence of this portion of c-myc mRNA showing
the locations of the five identified in vivo mRNA
degradation intermediates is shown in Fig. 8B. As with
albumin mRNA, each of these sites was determined by the sequence of
the junction between the ligated primer and the c-myc
mRNA degradation intermediate. Band 4 is particularly noteworthy here, because this corresponds to a previously mapped site
for in vitro cleavage of c-myc mRNA (8), thus
supporting the notion that the in vivo degradation of
c-myc mRNA involves endonucleolytic cleavage. In
addition, these results demonstrate that the LM-PCR approach to mapping
in vivo mRNA decay intermediates is generally applicable
to both highly abundant mRNAs like albumin, and rare mRNAs like
c-myc.
With few exceptions, mRNA degradation in vivo is
not accompanied by the appearance of stable degradation intermediates.
The most likely explanation for this is that in vivo these
intermediates are subject to rapid exonucleolytic clearance. However,
decay intermediates are observed using crude in vitro decay
systems, and the similarity seen between in vivo and
in vitro decay intermediates from the 3'-end of albumin
mRNA in Figs. 5 and 6 underscores the usefulness of such systems in
recapitulating steps in mRNA decay. Primer extension and S1
protection assays have been used successfully to identify in
vivo endonuclease cleavage of apo-very low density lipoprotein II
mRNA (7) and transferrin receptor mRNA (9), and we have used
these approaches to demonstrate in vivo cleavage within the
APyrUGA consensus PMR-1 cleavage sites in region A of albumin mRNA
both here and in a previous report (17). However, these techniques work
best with highly abundant mRNAs, and even with these its long
exposure times may be necessary to visualize some decay intermediates.
In this report we introduce ligation-mediated PCR for the rapid and
precise mapping of in vivo mRNA decay intermediates. The
advantages offered by LM-PCR include its ease of implementation, scalability, and ability to identify degradation intermediates from
even rare mRNAs such as c-myc. Although LM-PCR and
primer extension were used in this study to confirm the involvement of PMR-1 in the in vivo endonucleolytic degradation of albumin
mRNA, by definition, LM-PCR cannot distinguish between the 3'-end
of a decay product generated by endonuclease cleavage versus
a pausing site for a 3'-5'-exonuclease. That being said, Shyu and
coworkers3 (Department of
Biochemistry and Molecular Biology, University of Texas Medical Center,
Houston, TX) were unable to demonstrate in vivo exonuclease
pausing at poly(G) tracts as has been observed by Parker and coworkers
for yeast mRNA decay (27). This leads us to conclude that the decay
intermediates identified here, particularly for c-myc
mRNA, are the products of endonuclease cleavage.
Previous work from our laboratory identified PMR-1 as an
estrogen-induced endonuclease whose activity appeared on polysomes coincident with the estrogen-induced destabilization of albumin and
other serum protein mRNAs (14). Using antibodies to PMR-1 we
recently found that this mRNA endonuclease resides on polysomes in
a latent form that can be released with EDTA as part of a >670-kDa mRNP complex.2 Estrogen induces a 22-fold increase in unit
activity of polysome-bound PMR-1, which is accompanied by the
coordinate disappearance of both albumin mRNA and PMR-1 from
polysomes.2 The identification of estrogen-induced in
vivo decay intermediates in Figs. 3 and 4 corresponding to
products of PMR-1 cleavage lends considerable support to a central role
for PMR-1 in catalyzing the destabilization of albumin mRNA.
We recently demonstrated that vitellogenin mRNA, which is induced
and stabilized by estrogen, contains two APyrUGA elements in the 3'-UTR
that are cleaved by PMR-1. The vitellogenin mRNA 3'-UTR is bound by
vigilin, a 155-kDa estrogen-induced multi-KH domain protein (28), and
this binding inhibits in vitro cleavage by PMR-1 (25). High
affinity binding by vigilin requires a relatively unstructured target
sequence, and although albumin mRNA region A contains the same
APyrUGA PMR-1 cleavage sites as the vitellogenin mRNA 3'-UTR,
vigilin binds poorly to this highly structured sequence and is unable
to protect it from PMR-1 cleavage. Results presented here show
significantly greater in vitro and in vivo
cleavage within the paired APyrUGA sites in the single-stranded loop of albumin mRNA region A than within the same paired element in a hairpin structure in region G. Together these data point to the interplay between primary sequence and secondary structure of an
mRNA target both for determining susceptibility to cleavage by an
mRNA endonuclease and for mRNA stabilization resulting from protein binding to these sites.
Finally, we examined the degradation of c-myc mRNA as a
proof of principle for the applicability of the LM-PCR approach to the
identification of in vivo mRNA decay intermediates. The
experiment in Fig. 8 focused on the coding region determinant in
c-myc mRNA that has been extensively characterized
in vitro by Ross and coworkers as a site for ribosome
pausing, endonuclease cleavage, and protection from cleavage by the
binding of a KH-domain protein (8, 18, 29, 30), but whose cleavage
in vivo had yet to be demonstrated. Our results identified
degradation intermediates in this region, one of which (site 4)
corresponds to the major site for in vitro cleavage by a
polysome-associated endonuclease (8). These results lend further
validity to the use of in vitro systems for analyzing the
biochemistry of mRNA decay and underscore the general applicability of LM-PCR to the identification of in vivo decay intermediates.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin (11). Estrogen induces a global change in the profile of
proteins expressed in Xenopus liver resulting from the
coordinate destabilization of the serum protein mRNAs (12) and the
transcriptional induction and stabilization of the mRNA for the
yolk protein precursor vitellogenin (13). mRNA destabilization in
this tissue is mediated through the activation of polysomal
ribonuclease 1 (PMR-1)1 (14),
a novel endonuclease related to the peroxidase gene family (15). PMR-1
is a component of a large mRNP complex bearing its substrate mRNAs,
and mRNA decay is initiated by a 22-fold increase in the unit
activity of the polysome-bound enzyme following estrogen stimulation.2 This global
change in mRNA decay occurs in the absence of major changes in the
amount of cellular PMR-1. The purpose of the present study was to
determine whether this increase in unit activity is matched by the
appearance of albumin mRNA decay intermediates characteristic of
endonuclease cleavage by PMR-1.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was injected in 0.1 ml of a solution of 10%
Me2SO/90% propylene glycol into the dorsal lymph sac.
Animals were anesthetized with 0.1% tricaine methanesulfonate prior to
removing the liver.
-mercaptoethanol, and the mixture was homogenized at
4 °C for 2 min at 4000 rpm using a Teflon glass homogenizer. The
homogenate was centrifuged at 12,000 × g for 10 min at
4 °C to remove any insoluble material. One-tenth volume of 20%
(w/v) N-lauroylsarcosine was added, and the mixture was
heated at 65 °C for 2 min to denature protein. 0.1 g of CsCl/ml
liver extract was added, and the extract was layered over 9 ml of 5.7 M CsCl in a siliconized polyallomer tube and centrifuged
overnight at 113,000 × g at 22 °C in a Sorvall TH-641 swinging-bucket rotor. The supernatant was carefully removed, and the RNA pellet was dissolved in 3 ml of 5 mM EDTA,
0.5% (w/v) N-lauroylsarcosine, 5% (v/v)
-mercaptoethanol at 4 °C for 24 h. RNA was extracted once
with one volume of phenol:chloroform:isoamyl alcohol (25:24:1) and once
with one volume of chloroform:isoamyl alcohol (24:1). Sodium acetate,
pH 5.2, was added to 0.3 M and RNA was precipitated by
addition of 2.5 volumes of ice-cold ethanol. The recovered RNA pellet
was washed with 70% ethanol and dissolved in DEPC-treated water prior
to use.
80 °C for
30 min. The DNA pellet was recovered by centrifugation at 14,000 × g, 4 °C for 15 min, washed with cold 85% ethanol, and
dissolved in 12 µl of distilled H2O.
20 °C. The pellet was dissolved in 6 µl of gel
loading buffer II, and products were separated on a 6% denaturing polyacrylamide/urea gel and visualized by autoradiography. The positions of single-stranded G residues identified by RNase T1 digestion were used to model the secondary structure of this portion of
albumin mRNA using the MFOLD server (22).
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The LM-PCR protocol for identification of
mRNA decay intermediates. The LM-PCR protocol is based on the
observation that PMR-1 decay intermediates have 3'-hydroxyls. A DNA
ligation primer bearing a 5'-phosphate and a 3'-amino group are ligated
to a preparation of total RNA using RNA ligase. Reverse transcription
is primed with a complementary primer, and the resulting cDNA is
amplified using the complementary primer and a 5'-end-labeled primer
specific to the mRNA under study. Amplified products are separated
on a denaturing polyacrylamide gel, identified by autoradiography,
re-amplified by PCR, and sequenced to identify the 3'-end of the
degradation intermediate at the junction with the ligated primer.
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Fig. 2.
Consensus PMR-1 cleavage sites in albumin
mRNA. The 14 consensus APyrUGA PMR-1 cleavage sites in albumin
mRNA are indicated by vertical lines, with
brackets identifying the three sites with overlapping
elements. The 5' region A and 3' region G examined by LM-PCR are
indicated by the horizontal lines, with the locations of the
LM-PCR primers indicated by filled arrows. The open
arrow above the schematic indicates the position of the
oligonucleotide used for primer extension analysis in Fig. 4. The
albumin mRNA coding region is indicated in gray, and the
untranslated regions are indicated in black.
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Fig. 3.
LM-PCR identification of decay intermediates
in the 5'-coding region of albumin mRNA. A, LM-PCR
was performed with 10 µg of total lung RNA (lane 1) or 10 µg of total liver RNA prepared 0, 12, or 24 h after estrogen
administration using a 5'-32P-labeled primer complimentary
to a portion of the 5'-end of the mRNA. The PCR products were
separated on a 6% polyacrylamide/urea gel and visualized by
autoradiography. The numbers on the right side of
the autoradiogram correspond to the sites shown in C. B, the prominent band A11 was excised, re-amplified, and
sequenced to identify the junction between the ligated primer and the
mRNA degradation intermediate. C, each of the
numbered bands in A was re-amplified and
sequenced as in B, and their position is shown on the mapped
secondary structure of the 5'-coding region of albumin mRNA bearing
the overlapping APyrUGA elements whose in vitro cleavage by
PMR-1 was previously characterized (23). The open arrows
correspond to in vivo degradation intermediates that were
not observed by in vitro cleavage with purified PMR-1.
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Fig. 4.
Primer extension analysis of in
vivo and in vitro decay intermediates from
region A of albumin mRNA. A, primer extension was
performed on liver RNA isolated from control frogs (lane 1)
or animals injected estradiol 12 h prior to death (lane
2) using a 5'-32P-labeled primer whose position is
indicated by an open arrow above the schematic in Fig. 2.
Lanes 3-6 are a DNA sequencing ladder prepared using the
same primer and the corresponding albumin cDNA. The positions of
the in vivo decay intermediates corresponding to those
mapped onto the structure in Fig. 3C are indicated on the
left side of the autoradiogram. B, the RNA from
12-h estrogen-treated frogs was incubated for 30 min at 22 °C with
(lane 1) or without (lane 2) 40 units of purified
PMR-1. One unit of PMR-1 activity equals the amount of enzyme that
completely degrades 7 fmol of an albumin mRNA substrate transcript
corresponding to region A in 30 min at 22 °C. The positions of the
decay intermediates are indicated as in A.
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Fig. 5.
LM-PCR identification of decay intermediates
in the 3'-end of albumin mRNA. A, LM-PCR was
performed with 2 µg of total liver RNA from a 24-h estrogen-treated
frog using a primer that amplifies from position 1690 to the end of
albumin mRNA. The PCR products were separated on a 6%
polyacrylamide/urea gel and the bands of interest, shown by the
arrows, were extracted from the gel and sequenced. A 10-bp
molecular size ladder is shown in lane 1 (M).
B, the sequence of the 3'-end of albumin mRNA
corresponding to the region analyzed in A is shown with the
in vivo degradation intermediates indicated with
arrows. Consensus APyrUGA sites are
underlined.
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Fig. 6.
In vitro cleavage of the 3'-end of
albumin mRNA. A, a 5'-end-labeled transcript,
corresponding to the 3' 310 nt of albumin mRNA analyzed in Fig. 4,
was incubated at 23 °C for the indicated times with 10 µg of
polysome extract from 24-h estrogen-treated frogs prepared as described
previously (14). The products were separated on a 6%
polyacrylamide/urea gel and visualized by autoradiography, and the
degradation fragments corresponding to the in vivo cleavage
products are indicated with arrows. As in Fig. 3 cleavage
within the APyrUGA elements is identified by open arrows.
B, the transcript used in A was incubated for the
indicated times with 20 units of purified PMR-1. One unit of PMR-1 is
the amount needed to completely cleave 7 fmol of albumin substrate
transcript in 30 min at 23 °C. The products were separated as
described in A and are labeled corresponding to the in
vivo cleavage sites on the right.
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Fig. 7.
Secondary structure of the 3'-end of albumin
mRNA. The 5'-32P-labeled transcript for the
albumin mRNA 3'-end was incubated with 5 units of RNase T1, and the
products were separated on a denaturing 6% polyacrylamide/urea gel.
Lanes 1 and 2 correspond to 10 and 15 min of digestion. The
positions of cleaved G residues (identified by dots on the
autoradiogram) were determined by mobility relative to size standards.
These were used to guide the generation of a predicted secondary
structure using MFOLD. The RNase T1 cleavage sites appear as
H in the structure, and the eight cleavage sites mapped
in vivo and in vitro are labeled by the
arrows. The four APyUGA sequences present are identified by
the boxes. Note that the APyrUGA site in the bottom
left portion of the structure was not resolved in the gels used in
Figs. 5-7.
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Fig. 8.
LM-PCR mapping of in vitro
degradation intermediates within the c-myc
coding region determinant. A, LM-PCR was
performed as in Fig. 3 with 2 µg of total RNA isolated for MEL cells
using a primer specific to a region upstream of the c-myc
CRD. The PCR products were separated on a 6% acrylamide/urea gel, and
the bands of interest, shown by the arrows, were excised out
and sequenced. B, the region of c-myc mRNA
containing the CRD is shown with the arrows indicating the
locations of the in vivo degradation intermediates
identified in A. The underlined region
corresponds to the region previously shown to be cleaved in
vitro by a polysome-associated endonuclease (8).
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Elena Chernokalskaya for her help with the primer extension experiments in this study.
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FOOTNOTES |
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* This work was supported by Grant GM38277 from NIGMS, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biochemistry, The Ohio State University, 1645 Neil Ave., Columbus, OH 43210-1218. Tel.: 614-688-3012; Fax: 614-292-7232; E-mail: schoenberg.3@osu.edu.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M010483200
2 Cunningham, K. S., Hanson, M. N., and Schoenberg, D. R. (2001) Nucleic Acids Res. 29, 1156-1162.
3 A.-B. Shyu, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: PMR-1, polysomal ribonuclease 1; RT-PCR, reverse transcription-polymerase chain reaction; LM-PCR, ligation-mediated PCR; MEL cells, murine erythroleukemia cells; DTT, dithiothreitol; BSA, bovine serum albumin; nt, nucleotide(s); UTR, untranslated repeat; DEPC, diethylpyrocarbonate; KH, hnRNPK homology; mRNP, messenger ribonucleoprotein.
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