From the Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1064
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ABSTRACT |
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Many mRNAs in mammalian cells decay via a
sequential pathway involving rapid conversion of polyadenylated
molecules to a poly(A)-deficient state followed by rapid degradation of
the poly(A)-deficient molecules. However, the rapidity of this latter
step(s) has precluded further analyses of the decay pathways involved.
Decay intermediates derived from degradation of poly(A)-deficient
molecules could offer clues regarding decay pathways, but these
intermediates have not been readily detected. Cell-free mRNA decay
systems have proven useful in analyses of decay pathways because decay
intermediates are rather stable in vitro. Cell-free systems
indicate that many mRNAs decay by a sequential 3'-5' pathway
because 3'-terminal decay intermediates form following deadenylation.
However, if 3'-terminal, in vitro decay intermediates
reflect a biologically significant aspect of mRNA turnover, then
similar intermediates should be present in cells. Here, I have compared
the in vivo and in vitro decay of mRNA
encoded by the c-myc proto-oncogene. Its decay both in vivo and in vitro occurs by rapid removal of
the poly(A) tract and generation of a 3'-terminal decay intermediate.
These data strongly suggest that a 3'-5' pathway contributes to
turnover of c-myc mRNA in cells. It is likely that
3'-5' decay represents a major turnover pathway in mammalian cells.
The steady-state levels of mRNAs depend upon their combined
rates of synthesis and processing in the nucleus, transport from the
nucleus to cytoplasm, and decay in the cytoplasm. An early step in the
cytoplasmic decay of many mRNAs in eukaryotes is exoribonucleolytic shortening of the poly(A) tail (reviewed in Refs. 1-4). In the yeast
Saccharomyces cerevisiae, the 5'-3' and 3'-5' pathways
appear to be the two major pathways for mRNA decay. In both
pathways, a poly(A) nuclease shortens the poly(A) tract to 10-15
nt.1 In the 5'-3' pathway,
the enzyme Dcp1p then removes the 5' cap structure, and the Xrn1p
exoribonuclease degrades the mRNA 5'-3' (2, 5-7). In the 3'-5'
pathway, decay of the 10-nt poly(A) tract continues after the initial
phase of deadenylation. Degradation within the 3'-UTR then ensues 3'-5'
via the exosome, a heteropentameric protein complex (6, 7). Yeast
mRNAs also decay by alternate pathways under some circumstances.
For example, some mRNAs decay by an endonucleolytic cleavage event
that is the rate-limiting step in their turnover because the cleavage
is independent of poly(A) shortening (2). Additionally, mRNAs
containing nonsense codons decay by a pathway that involves
deadenylation-independent decay processes (8, 9).
By contrast, decay pathways in mammalian cells are not as well
understood. Like yeast mRNAs, many mammalian mRNAs decay
initially by exoribonucleolytic shortening of the poly(A) tract.
However, subsequent decay steps involving the mRNA body generally
occur so quickly that decay intermediates are not detected, thus
precluding further analyses of decay pathways. Nonetheless, some recent
indirect evidence suggests that mammalian mRNAs may decay by a
5'-3' pathway. For example, a polymerase chain reaction-based analysis
involving simultaneous comparisons of poly(A) tract lengths and
presence of a cap structure revealed that only mRNAs with short
poly(A) tracts lacked a cap structure (10). This suggests that
decapping of mammalian mRNAs occurs once deadenylation activities
shorten the poly(A) tract to some critical length. Additional evidence for a 5'-3' decay pathway in mammalian cells is the identification of a
murine XRN1 homologue that functions in S. cerevisiae (11). Together, these data suggest that 5'-3' mRNA decay processes may be
conserved in eukaryotes.
A 3' to 5' decay pathway for mammalian, polyadenylated mRNAs is
also likely for several reasons. (i) HeLa cells, a human cervical carcinoma cell line, contain a complex homologous to the yeast exosome
(6). (ii) Human H4 histone mRNA, which lacks a poly(A) tract,
decays 3'-5' in cells (12). Degradation is likely because of an
exoribonuclease that pauses within the 3'-terminal stem-loop structure,
generating progressively shorter decay intermediates that lack 5 nt,
then 12 nt, from the 3' end (13). (iii) In vitro mRNA
decay extracts prepared from mammalian cells degrade labile mRNAs
3'-5' by rapid deadenylation followed by generation of 3'-terminal decay intermediates (reviewed in Refs. 14 and 15). The same enzymes
operative in vivo presumably generate these intermediates in vitro. However, detection of such mRNA decay
intermediates in cells has proven difficult (1, 14). Thus, an
unanswered question is whether 3'-5' decay of polyadenylated mRNAs
occurs in mammalian cells as indicated by 3'-terminal decay
intermediates. Here, I have addressed this question by comparing the
decay of c-myc mRNA in vitro and in cells.
c-myc mRNA, which encodes the Myc transcription factor
and oncoprotein, is a labile, polyadenylated transcript (reviewed in
Ref. 16). In vitro, it rapidly decays by a sequential
pathway involving rapid deadenylation to a deadenylated or
oligoadenylated form; this is followed by degradation of the mRNA
body generating easily observed 3'-terminal decay intermediates. Its
decay then continues in a 3'-5' direction (17). c-myc
mRNA decays by rapid deadenylation in vivo as well. Also
observed is a 3'-terminal decay intermediate that appears to have a
similar 3' end as an intermediate generated during in vitro
decay. These data provide strong evidence that a 3'-5' pathway
contributes to decay of c-myc mRNA, and perhaps other
polyadenylated mRNAs, in mammalian cells.
Restriction enzymes and RNasin were obtained from Promega Corp.
(Madison, WI). RNase H, oligo(dT)12-18, and
oligo(dT)-cellulose were from Amersham Pharmacia Biotech. Creatine
phosphate, creatine phosphokinase, and actinomycin D were from
Calbiochem (La Jolla, CA). [ Actinomycin D Treatment of Cells, Preparation of RNA, and RNase H
Analysis--
Exponentially growing K562 cells, a human
erythroleukemia-like cell line (18, 19), were cultured with 5 µg/ml
actinomycin D for various lengths of time at 37 °C. For each time
point, cells were harvested and total RNA was prepared by lysis of
cells, phenol extraction of proteins, and pelleting of RNA through a
pad of CsCl as described (12). RNA concentrations were determined
spectrophotometrically by absorption at 260 nm. Ten micrograms of each
RNA sample were subjected to oligonucleotide-directed RNase H cleavage
using an antisense c-myc oligodeoxynucleotide
(5'-CAAGTTCATAGGTGATTGCTG-3') which anneals approximately 400 nt
upstream of poly(A) site 2 (17). Deadenylated RNAs were prepared
in vitro by incubating the time-zero RNA sample with
oligo(dT)12-18 and RNase H. The RNA samples were
fractionated in a denaturing agarose gel and blotted to a membrane. The
Oligo(dT)-cellulose Chromatography of Cellular RNA--
Ten
milligrams of RNA purified from 5 × 108 exponentially
growing K562 cells was heat-denatured and loaded onto a column
containing 400 mg of oligo(dT)-cellulose in column binding buffer (10 mM Tris-HCl, pH 8.1, 0.5 M sodium chloride, 1 mM EDTA, 0.1% sodium dodecyl sulfate). The flow-through
fraction was loaded onto the column a second time. The column was then
washed with 10 ml of column binding buffer. RNA bound to the column was
eluted with 10 ml of column elution buffer (10 mM Tris-HCl,
pH 7.5, 1 mM EDTA, 0.05% sodium dodecyl sulfate). The
flow-through fraction and washes were pooled and defined as the unbound
fraction. RNA from both the unbound fraction (i.e.
poly(A Radiolabeling of Probes--
The same c-myc probe was
used for both RNase protection assays and for the RNA blot (described
above). It was prepared by in vitro transcription of
SspI-digested plasmid pSP65myc(CLARI) (17) using SP6 RNA
polymerase and [ RNase Protection Assay and Nuclease S1
Mapping--
c-myc mRNA was analyzed by an RNase P1+T1
protection assay utilizing a 620-nt probe spanning the 3'-terminal 210 nt of the mRNA as described (17). Human H4 histone and Preparation of Cellular Extracts--
Polysomes and the
130,000 × g postribosomal supernatant (S130) were
prepared from K562 cells using Buffer A (10 mM Tris-HCl (pH
7.6), 1 mM magnesium acetate, 1.5 mM potassium
acetate, 2 mM dithiothreitol, 1 µg each of leupeptin and
pepstatin A per ml, 0.1 mM phenylmethylsulfonyl fluoride)
as described (17).
In Vitro mRNA Decay Reactions--
In vitro
mRNA decay reactions were incubated at 37 °C in 50-µl
reactions containing 1.4 A260 units of polysomes
in a buffer containing 10 mM Tris-HCl (pH 7.6), 5 mM magnesium acetate, 100 mM potassium acetate,
2 mM dithiothreitol, 10 mM creatine phosphate, 0.7 units of creatine phosphokinase, 1 mM ATP, 0.4 mM GTP, 0.1 mM spermine, and 40 units of
RNasin. Some reactions were supplemented with 300 µg of S130
proteins. RNA was purified for each time point, and c-myc
mRNA was analyzed by an RNase P1+T1 protection assay as described above.
Cell-free mRNA decay systems degrade mRNAs via a 3'-5'
pathway involving gradual removal of the poly(A) tract followed by 3'-terminal degradation of the mRNA body. To examine the decay pathway of c-myc mRNA in cells, exponentially growing
K562 cells were cultured in the presence of actinomycin D to inhibit
new transcription. At various time points, cells were harvested for purification of total RNA. Decay of c-myc mRNA was
investigated by a modified Northern blot procedure that involves
oligonucleotide-directed RNase H cleavage of c-myc mRNA
in purified RNA samples, prior to the gel electrophoresis and blotting
steps. c-myc mRNA is cleaved approximately 400 nt from
its 3' end by this procedure Thus, the RNase H mapping assay permits
examination of 3'-end decay at a resolution higher than that allowed by
traditional Northern blotting (17, 20, 21). Fig.
1 shows the results of this analysis. The
time-zero point showed a heterogeneous smear (Fig. 1, top panel, lane 1). Additional treatment of the time-zero
RNA with oligo(dT)12-18 and RNase H reduced this smear to
a single band (top panel, lane 8). This indicated
that the smear was because of heterogeneous lengths of poly(A) tracts
within the population of cellular c-myc mRNA molecules.
With increasing time following inhibition of transcription, the poly(A)
tracts overall became shorter. By 90 min, most c-myc
mRNA molecules were completely deadenylated or had short adenylate
tracts (Fig. 1, top panel; compare lane 6 with
lane 8). Rapid deadenylation of c-myc mRNA was specific because the poly(A) tract of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP and
[
-32P]dCTP were from ICN Biomedicals (Irvine, CA).
Oligodeoxynucleotide synthesis was performed by Operon Technologies
(Alameda, CA). All other reagents were molecular biology grade.
-globin mRNA or the 3'-end of c-myc mRNA was
detected by incubating the membrane with the respective 32P-labeled probes, washing the membrane, and exposing it
to x-ray film as described (17). This RNase H mapping procedure permits high resolution analysis of cleavage products at the 3' end of c-myc mRNA (20, 21).
) RNA) and the eluted fraction (i.e.
poly(A+) RNA) was precipitated with ethanol, recovered by
centrifugation, and quantified spectrophotometrically by absorption at
260 nm.
-32P]UTP (>800 Ci/mmol). The probe
used for detection of human
-globin mRNA on the RNA blot was
prepared by radiolabeling plasmid pJW151 (containing the human
-globin cDNA; gift from J. Ross) by the random-primer method
using [
-32P]dCTP (>3,000 Ci/mmol) (22). A
radiolabeled probe for detection of human H4 histone mRNA by
nuclease S1 mapping was prepared by 3'-end labeling of plasmid pHh4A
digested with NcoI as described (12). A radiolabeled probe
for detection of human
-globin mRNA by nuclease S1 mapping was
prepared by 3'-end labeling of plasmid pDCY2 digested with
EcoRI as described (17).
-globin
mRNAs were analyzed by nuclease S1 mapping using 3'-end
radiolabeled probes as described (12, 17). Bands were quantified by
laser densitometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin was not rapidly shortened, and the mRNA was stable over the 3-h time course (Fig. 1, lower panel). Thus, c-myc mRNA was rapidly
deadenylated in cells generating deadenylated or oligoadenylated
mRNA molecules. These results are consistent with those from
earlier studies showing rapid deadenylation of c-myc
mRNA in cell-free decay reactions (17).
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Fig. 1.
Deadenylation of c-myc
mRNA in vivo. K562 cells were
cultured in 5 µg/ml actinomycin D for the indicated times. RNAs were
extracted and subjected to deoxyoligonucleotide-directed RNase H
mapping analysis for c-myc mRNA as described under
"Experimental Procedures." RNA samples were fractionated in an
agarose-formaldehyde gel, blotted to a membrane, hybridized to a probe
specific for the c-myc 3'-UTR, and exposed to x-ray film
(top panel). For comparison, the gel was loaded with a
sample of RNA that had also been treated with
oligo(dT)12-18 and RNase H to remove poly(A) tracts.
Lane M contains 32P-labeled marker
RNAs prepared by in vitro transcription; nucleotide lengths
are denoted to the right. Following decay of the signal for
c-myc, the blot was reprobed for -globin mRNA
(bottom panel).
Decay of c-myc mRNA in cell-free extracts also results
in the formation of abundant decay products mapping to the 3'-UTR (17). By contrast, none were obvious in the RNA blot analysis of in vivo decay shown in Fig. 1. However, it was likely that any
putative 3'-terminal decay products would be very unstable in cells and thus be low in abundance (1, 14). Therefore, the RNA samples utilized
in Fig. 1 were analyzed for possible 3'-terminal decay products by a
sensitive RNase P1+T1 protection assay. A 620-nt radiolabeled RNA probe
complementary to the 3'-terminal 210 nt of c-myc mRNA
was employed for this assay (Fig.
2A). The time-zero RNA
generated four protected fragments corresponding to intact c-myc mRNA molecules polyadenylated at four closely
spaced sites, referred to as poly(A) site 2 (17). These bands
diminished in intensity at approximately equivalent rates during the
time course. Quantitation of band intensities as a function of time
indicated a half-life of 45 min for c-myc mRNA. Also
observed was a very faint protected fragment that was shorter than the
four full-length c-myc mRNA species; it also decreased
in intensity with time (Fig. 2A, arrow). A 14-day
overexposure of the gel enhanced its intensity especially at later time
points (Fig. 2B, arrow). This band was not an
artifact of the RNase-digested probe because the control incubation of
probe with tRNA did not produce a band at this location in the gel
(Fig. 2B, lane 1). Additionally, this band was
not detected in a hybridization of the probe with full-length, in vitro synthesized c-myc mRNA, even though the
protected fragment corresponding to full-length mRNA was
over-exposed (Fig. 2C, compare lane 3 to
lane 2). These data suggested that the faint, protected fragment corresponded to a low abundance, 3'-terminal product of
c-myc mRNA decay in cells.
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If this band represents a decay intermediate generated following
deadenylation, then it should be detectable in a poly(A)
fraction of RNA from K562 cells and not be detectable in a
poly(A+) fraction. RNA was fractionated by chromatography
using oligo(dT)-cellulose (see "Experimental Procedures"). Analysis
of the RNA samples by agarose gel electrophoresis indicated efficient
separation of ribosomal RNA (Fig. 3A). As
a further control for the separation of poly(A
) and
poly(A+) RNAs, fractions were analyzed for the distribution
of H4 histone mRNA, which lacks a poly(A) tract;
-globin
mRNA, which is a polyadenylated mRNA with a half-life of
20 h; and c-myc mRNA, which is a labile, polyadenylated mRNA. Levels of H4 histone and
-globin mRNAs
were measured by nuclease S1 mapping assays. Quantitation of the
protected fragments and comparison of signals to the signal present in
total RNA indicated that all of the H4 histone mRNA was detected in the poly(A
) fraction; none was detected in the
poly(A+) fraction (Fig. 3B, lanes
3-5). As expected, most (80%) of the
-globin mRNA was
present in the poly(A+) fraction (Fig. 3B,
lanes 7-9). These analyses indicated a suitable separation
of poly(A
) and poly(A+) RNAs by
oligo(dT)-cellulose chromatography. The distribution of
c-myc mRNA was examined by RNase P1+T1 protection assay
of the fractionated RNAs. Sixty percent of c-myc mRNA
was present in the poly(A
) fraction and 40% was in the
poly(A+) fraction (Fig. 3C), indicating that a
substantial fraction of the full-length mRNA is likely
deadenylated. A 14-day overexposure of the gel showed the presence of
the band corresponding to the decay intermediate in the
poly(A
) fraction while none was detectable in the
poly(A+) fraction (Fig. 3D, compare lane
3, arrow with lane 4). These data further
suggested that the faint, protected fragment observed in RNase
protection assays of cellular RNA is a decay intermediate of
c-myc mRNA.
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Additionally, the results of the cellular decay experiments were
compared with those from cell-free decay reactions, where decay
products are readily observed (17). RNase P1+T1 protection analysis of
c-myc mRNA in decay reactions containing polysomes revealed a major protected fragment with a size similar to the one
observed during in vivo decay (Fig.
4A, band I). (The
faint, minor band above product I is not consistently observed.) A
smaller, less abundant fragment not readily detected in cellular RNA
was also observed (Fig. 4A, band II). Bands I and
II increased in abundance during incubation times up to 60 min, after
which they decreased in abundance. These results were consistent with
their being transient products of 3'-terminal decay in
vitro. Decay product I was also present in a trace amount in
time-zero RNA. This was not unexpected because c-myc
mRNA molecules should be undergoing decay in K562 cells at the time
of cell lysis. As shown in earlier studies, 3'-terminal decay products
form in vitro following removal of most, if not all, of the
poly(A) tract (17). Like the 3'-terminal decay product in cells,
in vitro decay products also fractionate with
poly(A) RNA by oligo(dT)-cellulose
chromatography.2
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The stabilities of some mRNAs, such as c-myc, are regulated by cytosolic factors, such as the S130 fraction, not associated with polysomes (23). Thus, formation of 3'-terminal decay intermediates was compared in cell-free reactions containing polysomes alone versus reactions containing polysomes and S130 proteins. In the S130-supplemented reactions, the abundance of decay product I was reduced compared with reactions lacking S130 (Fig. 4B, compare lanes 2-8 with lanes 9-11). This result was consistent with the ability of S130 components to accelerate 3'-terminal decay of c-myc mRNA (23, 24). Moreover, the low abundance of decay product I in S130-supplemented reactions reflected the low abundance of the 3'-terminal decay intermediate observed in cells (compare Fig. 4B, lanes 2-8 to Fig. 2). Decay product II, observed in cell-free reactions containing polysomes without S130, was not readily apparent in either S130-supplemented decay reactions (Fig. 4B, compare lane 11 to lanes 2-8) or in vivo (see Fig. 2). This could be because of its low abundance in these two cases. Altogether, comparison of the in vivo data with the in vitro data strongly argues that decay of c-myc mRNA in cells involves rapid deadenylation and generation of a low abundance, 3'-terminal decay intermediate.
Additionally, size comparison of the 3'-terminal decay product in
cellular RNA and decay product I in cell-free reactions suggested they
were the same (Fig. 4B, compare lane 13 to
lanes 2-11). Therefore, an RNase protection assay was
performed with RNA prepared from K562 cells and RNA prepared from
in vitro decay reactions (with and without added S130
proteins). These were run in a gel side by side with a DNA sequencing
ladder. Fig. 5 shows that in each RNA
sample, the 3' ends of the decay products mapped within one or two nt
of each other near the 3' termini of the four full-length
c-myc mRNA species. These results strongly suggest that
the 3'-terminal decay product I generated in vitro
represents molecules present in cells as well.
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DISCUSSION |
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In vitro, c-myc mRNA decays via a
sequential pathway involving rapid conversion of polyadenylated
mRNA molecules to a deadenylated or perhaps an oligoadenylated
form. Subsequently, 3'-terminal degradation within the 3'-UTR occurs
(17). Thus, c-myc mRNA clearly decays in
vitro by a 3'-5' pathway (see also Ref. 25). If this pathway is
biologically significant, then equivalent decay intermediates should be
present in cells. The data presented here indicate that decay of
c-myc mRNA in cells involves rapid deadenylation to a
deadenylated or perhaps an oligoadenylated form (Fig. 1). A low
abundance, 3'-terminal product of c-myc mRNA decay is
present in cells (Fig. 2). The product is only observed in a
poly(A) fraction of cellular RNA prepared by
oligo(dT)-cellulose chromatography (Fig. 3). This observation strongly
suggests that the product is likely a bona fide mRNA
decay intermediate rather than a minor cleavage/polyadenylation site.
Additional support for this conclusion comes from the finding that a
similar 3'-terminal decay product is observed in cell-free mRNA
decay reactions (Fig. 4). Finally, both the in vitro and
in vivo decay intermediates map to a similar location near
the 3' terminus of the 3'-UTR (Fig. 5). Taken together, these data are
consistent with the conclusion that a 3'-5' decay pathway contributes
to c-myc mRNA turnover in cells.
The mRNA decay intermediates generated in vivo and in vitro (particularly in S130-supplemented reactions) appeared similar. This reflects the fidelity of the in vitro system for reconstituting physiologically significant mRNA decay processes involving polyadenylated mRNAs (14, 15). This has obvious consequences for studies designed to purify the relevant mRNA decay factors. For example, in addition to the purification of deadenylating ribonucleases (e.g. DAN, Ref. 26), it will be important to identify and purify enzymes that exhibit 3'-5' ribonuclease activity with 3'-UTRs from labile mRNAs such as c-myc. These could possibly be one or more exoribonucleases given that the exosome, a complex of exoribonucleases responsible for 3'-5' decay in yeast, is also found in mammalian cells (6, 7). Moreover, both in vivo and in vitro decay of c-myc mRNA generates 3'-terminal decay intermediates. Thus, any putative ribonucleases obtained by purification should reconstitute the formation of the same decay intermediates observed in vivo if they are the authentic enzymes operative in cells. For instance, an exoribonuclease purified from ribosomal salt wash degrades H4 histone mRNA in vitro by generating the same decay intermediates observed in vivo (13). This provides compelling evidence that the histone exoribonuclease is responsible for decay in vivo.
A 3'-5' decay pathway does not exclude the possibility that other pathways could also be operative in cells. In fact, c-myc mRNA decays by an alternative pathway in some circumstances. For example, there is evidence that in differentiating cells c-myc mRNA decays directly without prior conversion to a deadenylated form (27). This may involve inactivation of the c-myc mRNA-binding protein CRD-BP and subsequent unmasking of an endoribonuclease site within the 3'-coding region (28-30). Additionally, the action of the endoribonuclease apparently does not require prior removal of the poly(A) tract. Thus, endoribonucleolytic cleavage of the coding region, rather than poly(A) shortening, may be the rate-limiting step in c-myc mRNA decay during differentiation.
Additionally, a 3'-5' pathway does not exclude a 5'-3' pathway acting simultaneously. In the yeast S. cerevisiae, some mRNAs decay by both 5'-3' and 3'-5' pathways (7). It is likely that c-myc mRNA decays in vivo by both pathways as well. This is based upon the observation that nuclease protection assays employing probes specific for either the 5' or 3' end indicated little difference in the measured half-life.2 By contrast, H4 histone mRNA decays predominantly 3'-5' (12). In any event, a possible advantage to the cell for having multiple pathways to degrade mRNAs would be to provide redundancy in the event one pathway were to become inactivated because of mutation, for example. In this case, the additional mRNA turnover pathway(s) could function. Consistent with the necessity of mRNA turnover processes is the demonstration that they are essential for viability of both yeast and bacteria. For instance, blocking both 5'-3' and 3'-5' decay pathways in yeast leads to inviability (7). Likewise, abating 3'-exoribonuclease activity in E. coli by mutation of both polynucleotide phosphorylase and RNase II leads to inviability (31). Messenger RNA decay processes will likely be essential for mammalian cells as well.
In summary, I have compared decay of c-myc mRNA in
vivo and in vitro and determined that similar decay
intermediates are generated in both systems. The data are consistent
with the conclusion that a 3'-5' decay pathway contributes to the
turnover of c-myc mRNA in vivo. Finally,
these data firmly establish the fidelity of the in vitro
mRNA decay system for 3'-5' decay of polyadenylated mRNAs.
Future studies will seek to identify, purify, and characterize the
relevant activities to reconstitute mRNA decay from purified components.
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ACKNOWLEDGEMENT |
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I thank Gerald Wilson for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Grant CA52443 from the National Institutes of Health (to G. B.).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. Tel.: 336-716-6756;
Fax: 336-716-9928; E-mail: gbrewer{at}wfubmc.edu.
2 G. Brewer, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: nt, nucleotide(s); RNase, ribonuclease; UTR, untranslated region.
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