(Received for publication, June 22, 1995)
From the
In an exploration of the molecular basis of cyclic AMP-induced
stabilization of Na/glucose cotransporter mRNA (SGLT1
isoform) accompanying cell differentiation in the pig kidney cell line
LLC-PK
, we have identified a 48-kDa cytoplasmic protein
factor, designated SG-URBP, which specifically binds a 120-nucleotide
sequence within the 3`-untranslated region of the SGLT1 message. A
46-nucleotide uridine-rich element within this region appears necessary
for specific binding, and the presence of the 3`-untranslated region is
necessary for message stabilization by cyclic AMP. The binding activity
of SG-URBP is up-regulated after cyclic AMP elevation and protein
kinase A activation, whereas protein dephosphorylation either in
vivo or in vitro is associated with loss of binding
activity. The increase in SG-URBP binding activity correlates with an
increase in the half-life of the SGLT1 message, suggesting a cause and
effect relationship.
Glucose transport by mammalian cells is a highly regulated
process. Two structurally, functionally, and genetically distinct
classes of glucose transporters have been identified, the GLUT family
of facilitative glucose transporters (Gould and Bell, 1990) and the
SGLT family which mediates Na-coupled secondary active
transport of glucose. SGLT1 transporters are restricted in tissue
expression to the apical membranes of small intestine and renal
proximal tubule and have been mapped to human chromosome 22 (Wright,
1993). SGLT1 also transports galactose and has been identified as the
locus of the inherited defect glucose-galactose malabsorption (Turk et al., 1991).
Much of what is currently known about
regulation of the expression of the SGLT type of glucose transporter
has been derived from studies using the cell line LLC-PK,
derived from porcine renal proximal tubule (Amsler and Cook, 1982; Peng
and Lever, 1993). This cell line permits the reconstitution of a fully
functional polarized epithelium in vitro. The development of a
confluent monolayer is accompanied by the appearance of a number of
differentiated functions characteristic of this epithelium including
the formation of tight junctions and microvilli, vasopressin
responsiveness, transepithelial salt and water transport, several
brush-border marker enzyme activities, and apical membrane
Na
-coupled glucose transport activity (reviewed in
Lever (1989)). Elevation of intracellular levels of cyclic AMP or
treatment with the differentiation inducer hexamethylene bisacetamide
(HMBA), (
)greatly increased both the levels of expression
and rate of appearance of several of these differentiated functions
including Na
-coupled glucose transport (Amsler and
Cook, 1982; Yoneyama and Lever, 1984; Peng and Lever, 1993; Yet et
al., 1994). These agents appear to act by independent and
synergistic mechanisms (Peng and Lever, 1993).
SGLT1 transcripts of
2.2 and 3.9 kb, differing in the length of the 3`-untranslated region
(3`-UTR) are observed in LLC-PK cells as a result of
alternative cleavage and polyadenylation (Ohta et al., 1990).
We have recently demonstrated that the half-life of the larger
transcript is increased by more than 8-fold after cyclic AMP elevation,
indicating that post-transcriptional regulation exerts a major
influence on SGLT1 expression (Peng and Lever, 1995). The observation
that the 3`-UTR is necessary to observe this effect suggested that
sequences within this region may regulate message stability in response
to cyclic AMP.
In the present study, we extend these observations by
identifying a cytoplasmic M = 48,000
mRNA-binding protein, designated SG-URBP, which binds a cognate
sequence containing a uridine-rich element (URE) within the SGLT1 mRNA
3`-UTR. SG-URBP binding activity is greatly increased in extracts from
LLC-PK
cell cultures after treatment with cyclic
AMP-elevating agents and is dependent on protein phosphorylation.
SG-URBP appears to be a new member in the inventory of RNA-binding
proteins which have in common the specific recognition of uridine-rich
stability-determining elements in mRNA (Belasco and Brawerman, 1993;
Chen and Shyu, 1995) and the only one identified thus far in this
category which exhibits regulation by cyclic AMP-dependent protein
phosphorylation.
In order to map the SG-URBP binding site, a series of
deletions were constructed within pB1-3UTR2. Plasmid
p3UTR2435 containing a 435-bp SGLT1 3`-UTR segment (nucleotides
2263 to 2697) was generated by deleting a StuI/HindIII fragment from plasmid pB1-3UTR2.
Plasmid p3UTR2
5 was constructed by inserting a 1276-bp EcoO109I fragment from plasmid pB1-3UTR2 into
pBluescript II SK(-) vector at the EcoO109I site.
p3UTR2
6 was generated by deletion of a 174-bp BamHI/StyI fragment from plasmid p3UTR2
435.
Similarly, p3UTR2
7 was generated by deletion of a 443-bp BamHI/AvaII fragment from pB1-3UTR2.
p3UTR2
8 was generated by inserting a 953-bp HincII
fragment from plasmid pB1-3UTR2 into the pBluescript II
SK(-) vector at the HincII site.
Plasmids pT7/T3
-19-AUUUA and pT7/T3
-19 were gifts from Dr. J. S. Malter
(University of Wisconsin). Plasmids pT3ARE-WT1 and pUC-GAPDH13 were
kindly provided by Dr. A.-B. Shyu (University of Texas, Houston).
Plasmid pGAPDH486 was generated by deleting a 539-bp NsiI/EcoRV fragment from pBS-GAPDH13.
The following sense transcripts represent segments within the 3UTR2
region and also contain the same 69 nt of the pBluescript vector
sequence at the 5` end. 3UTR21, a 436-nt transcript synthesized
from HincII-linearized p3UTR2
435 containing 367 nt of the
pig SGLT1 3`-UTR(2263-2629); 3UTR2
2, a 375-nt transcript
synthesized from NspI-linearized p3UTR2
435 containing 306
nt of the pig SGLT1 3`-UTR(2263-2629); 3UTR2
3, a 313-nt
transcript synthesized from StyI-linearized p3UTR2
435
containing 244 nt of the pig SGLT1 3`-UTR(2263-2568);
3UTR2
4, a 220-nt transcript synthesized from EcoO109I-linearized p3UTR2
435 containing 151 nt of the
pig SGLT1 3`-UTR(2263-2413). Other transcripts utilized include:
3UTR2
5, a 397-nt transcript synthesized from StuI-linearized p3UTR2
5 containing 284 nt of the pig
SGLT1 3`-UTR(2345-2629) and 113 nt from pBluescript; 3UTR2
6,
a 179-nt transcript synthesized from HincII-linearized
p3UTR2
6 containing 123 nt of the pig SGLT1 3`-UTR(2440-2562)
and 56 nt from pBluescript; 3UTR2
7, a 176-nt transcript
synthesized from HincII-linearized p3UTR2
7 containing 120
nt of the pig SGLT1 3`-UTR(2510-2629) and 56 nt from pBluescript;
3UTR2
8, a 166-nt transcript synthesized from HincII-linearized p3UTR2
8 containing 68 nt of the pig
SGLT1 3`-UTR(2562-2629) and 98 nt from pBluescript; c-fos ARE, a 174-nt transcript synthesized from BglII-linearized pT3ARE-WT1 containing 84 nt of human
c-fos 3`-UTR and 80 nt of the rabbit
-globin coding
region with 10 nt from pT7/T3
-19 (Shyu et al., 1989);
AU80, a 80-nt transcript synthesized from EcoRI-linearized
pT3/T7
-19 containing 4 AUUUA repeats (Malter, 1989); T7/T3
-19
60, a 60-nt transcript synthesized from EcoRI-linearized pT7/T3
-19; GAPDH486, a 539-nt antisense
transcript synthesized from XbaI-linearized pBS-GAPDH486
containing 486 nt of rat glyceraldehyde-3-phosphate dehydrogenase
coding region and 53 nt from pBluescript SK(-).
Sense-strand
RNA transcripts were synthesized using T3 RNA polymerase (Stratagene)
with the exception of probes AU80, T3/T7 -19
60, and GAPDH486
which were transcribed using T7 RNA polymerase (Stratagene). After
transcription, RNase-free DNase (RQ1 DNase, Promega) was added and
mixtures were incubated for an additional 30 min at 37 °C to remove
template DNA. Then, 20 µg of yeast tRNA was added to each tube as
carrier, followed by dilution with diethylpropylcarbonate-treated water
to a final volume of 100 µl. Aliquots of 1 µl of each reaction
mixture were removed and further diluted with 99 µl of
diethylpropylcarbonate-treated water for trichloroacetic acid
precipitation (Maniatis et al., 1989) to calculate the amount
of cRNA produced. Labeled transcripts were extracted with
phenol/chloroform and precipitated twice with 2 M ammonium
acetate in 2.5 volumes of ice-cold ethanol at -80 °C for at
least 1 h. The final pellet was washed with 80% ice-cold ethanol,
air-dried, dissolved in diethylpropylcarbonate-treated water, stored at
-20 °C, and used as soon as possible. The integrity of the
transcripts was verified by agarose electrophoresis.
To synthesize
unlabeled transcripts in quantity for competition experiments,
transcription reactions were performed by the same procedure described
above except [P]UTP was replaced by 0.5 mM UTP, and the total volume of each reaction mixture was increased
to 100 µl.
Figure 1: Restriction map of SGLT1 3`-UTR cDNA. The region encompassed by plasmid pB1-3UTR1 is shown. Regions encoding the four sense RNA probes used in this study are indicated (3UTR1-4). The thin lines represent the sense SGLT1 RNA probes produced by in vitro transcription from plasmid constructs. A uridine-rich region (URE) and three AUUUA motifs are indicated in the diagram.
This difference was more clearly
apparent when RNA-protein incubations were UV-cross-linked after the
RNase T1 digestion step to generate covalent bonds between the P-labeled sense RNA probe and associated proteins. The
products were analyzed by SDS-PAGE in a label transfer experiment (Fig. 2). A 28-kDa RNA-protein complex was observed in samples
derived from control cultures (lane 2) whereas samples derived
from IBMX-induced cultures (lane 5) exhibited a 48-kDa
RNA-protein complex accompanied by a loss of the 28-kDa complex. The
presence of a 250
molar excess of specific competitor RNA
(unlabeled 3UTR2 RNA) effectively blocked the appearance of these
complexes (lanes 3 and 6), while addition of the same
amount of nonspecific competitor RNA (a 529-bp unlabeled antisense
glyceraldehyde-3-phosphate dehydrogenase RNA) had much less effect (lanes 4 and 7), indicating that these binding
activities are specific. The formation of the 48-kDa complex was
proportional to the amount of cellular extract added to the binding
reaction and abolished if the binding reaction mixture was treated with
proteinase K (Fig. 3). These results demonstrate the presence of
a trans-acting protein factor in LLC-PK
cells
which specifically interacts with the 3UTR2 mRNA fragment in response
to IBMX treatment.
Figure 2:
IBMX treatment induces a 48-kDa RNA
binding activity specific to 3UTR2 RNA. Radiolabeled sense RNA probe
3UTR2 was incubated with lysates from control (lanes
2-4) or IBMX-treated (lanes 5-7) LLC-PK cells in the absence (lanes 2 and 5) or
presence of a 250
molar excess of either specific competitor (SC, lanes 3 and 6) or nonspecific
competitor RNA (NC, lanes 4 and 7).
Following RNase T1 digestion, samples were UV-cross-linked and resolved
by 10% SDS-PAGE.
Figure 3:
Protein concentration dependence and
proteinase K sensitivity of 48-kDa complex formation. A, P-labeled 3UTR2 RNA was incubated with increasing amounts
of cytoplasmic extracts from either control (lanes 2-5)
or IBMX-treated (lanes 6-9) LLC-PK
cells and
analyzed by the UV-cross-linking assay. B, cytoplasmic lysates
were preincubated with the indicated amounts of proteinase K at 37
°C for 30 min prior to the UV-cross-linking
assay.
The formation of the 48-kDa complex was also
induced by other cAMP-elevating agents such as 8-bromo-cAMP, dibutyryl
cAMP, and forskolin (Fig. 4, lanes 4, 5, and 6). The IBMX-induced complex formation was reduced
substantially when LLC-PK cells were exposed to H-89 (lane 3), a specific protein kinase A inhibitor (Chijiwa et al., 1990). Addition of the protein kinase C inhibitor H-7
(Hidaka et al., 1984) to cells produced a marginal inhibitory
effect on the IBMX-induced 48-kDa complex (lane 8). These
results strongly suggest that protein kinase A is involved in the
regulation of the formation of the 48-kDa RNA-protein complex.
Figure 4:
The
48-kDa RNA binding activity is abrogated by the protein kinase A
inhibitor H-89. P-labeled 3UTR2 was incubated with
cytoplasmic lysates (40 µg) from untreated control cells or cells
treated for 4 days as indicated before UV-cross-linking assay. Lane
1, control; lane 2, 1 mM IBMX; lane 3,
1 mM IBMX plus 50 µM H-89; lane 4, 250
µM 8-bromo cAMP (8Br-cAMP); lane 5, 100
µM dibutyryl cAMP (dbcAMP); lane 6, 100
µM forskolin; lane 7, 5 mM HMBA; lane 8, 1 mM IBMX plus 40 µM H-7.
The differentiation inducer HMBA also promoted an increase in the half-life of the 3.9-kb SGLT1 message (Peng and Lever, 1995). HMBA, 5 mM, also induced an increase in the 48-kDa RNA-protein cross-linked complex (Fig. 4, lane 7) which was diminished by H-89 but not by H-7 (not shown), suggesting a possible role of cyclic AMP-protein kinase A in mediating this response.
Figure 5:
Phosphatase treatment prevents formation
of the 48-kDa RNA-protein complex. Potato acid phosphatase (PP) was preincubated with cytoplasmic lysates from
IBMX-treated LLC-PK cells in the presence of increasing
amounts of the phosphatase inhibitor microcystin LR (MICC-LR)
for 15 min before addition of
P-labeled 3UTR2 (1 ng) to
initiate the RNA-protein binding reaction. After UV-cross-linking, the
products were analyzed by 10% SDS-PAGE. The presence of MICC-LR
partially protected against inhibition by potato acid phosphatase of
48-kDa complex formation (lanes 6-8) compared with
extracts treated with potato acid phosphatase alone (lane 4).
MICC-LR by itself had no significant effect on the RNA binding activity (lane 5) compared to that in uninduced control (lane
2) or IBMX-induced cells (lane 3). Lane 1, free
P-labeled 3UTR2 probe without
lysate.
Figure 6: In vivo inhibition of phosphatases stimulates the formation of the 48-kDa complex in uninduced control cell extracts. Cytoplasmic lysates from control or IBMX-treated cells as indicated were prepared at the indicated times after addition of 2 µM okadaic acid (OA) and analyzed by UV-cross-linking using radiolabeled 3UTR2 (1 ng). Lane 1, free probe (FP).
Figure 7:
Effect of uridine-rich RNA competitors on
the formation of the 48-kDa complex. A cytoplasmic lysate (30 µg)
from IBMX-treated LLC-PK cells was preincubated for 10 min
with a 250
molar excess of the indicated unlabeled RNAs (lanes 3-10) prior to the addition of radiolabeled 3UTR2
RNA and UV-cross-linking assay. Lane 1, free
P-labeled 3UTR2 probe without lysate; lane 2, no
unlabeled RNA added. SC, specific competitor RNA (unlabeled
3UTR2 RNA).
To test whether AU80 or c-fos ARE RNA interacts with the
same protein factor that forms the 48-kDa complex with SGLT1 3UTR2,
cytoplasmic extracts from IBMX-treated LLC-PK cells were
incubated with radiolabeled AU80 or c-fos ARE RNA and then a
UV-cross-linking experiment was performed. As shown in Fig. 8,
in the case of AU80, 3 RNA-protein complexes, with apparent molecular
masses of 63, 54, and 40 kDa, respectively, were observed (lane
4). Interestingly, complexes of 63 and 54 kDa were also observed
to be cross-linked to SGLT1 probes 3UTR3 and 3UTR4 (not shown), which
contain AUUUA motifs (Fig. 1). The c-fos ARE RNA formed
a 42-kDa cross-linked complex (lane 5). However, none of these
RNA segments formed the 48-kDa complex observed with SGLT1 3UTR2 RNA (lane 3). Although the possibility exists that small
differences in apparent molecular mass of each complex may reflect a
contribution from different sizes of the residual RNA fragment
cross-linked in each case rather than differences in the identity of
the protein, this finding suggests that neither AU80 nor c-fos ARE RNAs contain the cognate sequence recognized by protein(s)
forming the 48-kDa complex with SGLT1 3UTR2.
Figure 8:
Specificity of the 48-kDa RNA binding
activity. Cytoplasmic lysates (30 µg) from IBMX-treated cells were
incubated with P-labeled 3UTR2, AU80, or c-fos ARE sense RNA probes (1 ng of each) as shown and analyzed by the
UV-cross-linking assay. Lane 1, free
P-labeled
3UTR2 probe without lysate; lane 2,
P-labeled
3UTR2 RNA incubated with control lysate.
Figure 9:
Mapping of the protein binding sites in
the 3`-UTR region of SGLT1 3UTR2 RNA. A series of P-labeled sense RNA probes representing different regions
of 3UTR2 (see Fig. 10) were transcribed and incubated with
cytoplasmic lysates. Lane 1, free
P-labeled 3UTR2
probe without lysate (FP); lanes 2-6, a set of
3`-deleted 3UTR2 probes incubated with lysates from IBMX-induced cells; lanes 7-14, a set of 5`-deleted 3UTR2 probes incubated
with lysates from uninduced control (lanes 7-10) or
IBMX-induced (lanes 11-14)
cells.
Figure 10:
Mapping of the cognate RNA sequence
involved in formation of the 48-kDa complex. A, restriction
map of the 3UTR2 region of pig kidney SGLT1. The open box represents 3UTR2 region of the SGLT1 clone. Thin lines represent the sense SGLT1 RNA probes. The location of the URE is
shown. B, summary of UV-cross-linking results using sense RNAs
covering various domains within this region. +++, strong
binding; +, weak binding; ±, very weak binding; -, no
detectable binding. C, nucleotide sequence of probe 3UTR7
which contains the U-rich element (underlined) and encompasses
the SG-URBP binding site. The arrow indicates the HincII site.
The mechanisms responsible for maintenance of the
differentiated phenotype in renal epithelial cells are poorly
understood. Expression in cell culture of the high affinity
Na/glucose cotransporter (SGLT1), a marker for late
proximal tubule (S3), is regulated by cell density, differentiation
inducers, as well as a number of agonists of intracellular signaling
pathways. We have recently demonstrated that post-transcriptional
stabilization of the SGLT1 message plays a prominent role in
differentiation-associated expression of this transporter induced by
either cyclic AMP or the differentiation inducer HMBA (Peng and Lever,
1995). Message stabilization required the presence of the 3`-UTR,
suggesting that cis-acting sequences in this region were
involved. This mechanism would permit the cell to increase rapidly its
commitment to synthesize a specialized end product of differentiation
in response to various extracellular signals.
Our present results
show that the 3`-UTR of porcine renal Na/glucose
cotransporter SGLT1 mRNA contains a U-rich region (URE) that exhibits a
specific, cyclic AMP-dependent and protein phosphorylation-dependent
interaction with a cytoplasmic protein of apparent M
= 48,000 which we have named SG-URBP. The simplest
hypothesis to explain our results is that SG-URBP is a phosphoprotein
in its activated state, and its binding affinity to SGLT1 mRNA is
directly activated by protein kinase A-mediated phosphorylation.
Alternately, protein kinase A may be indirectly involved via a kinase
cascade nor can we rule out the possibility that phosphorylation of
another protein may be involved in activation of SG-URBP. The active
phosphorylated form of SG-URBP would be hydrolyzed to an inactive (or
low affinity) dephosphorylated form by an unidentified protein
phosphatase. Our UV-cross-linking assay is only capable of assaying
SG-URBP in its activated, RNA binding state, and, in the absence of
specific antibodies, the putative inactive form cannot be measured.
Direct demonstration of the mechanism will require purification of
SG-URBP.
We have identified a 120-base region within the 3`-UTR of the renal SGLT1 message (Fig. 10C) which specifically interacts with SG-URBP to form the 48-kDa RNA-protein complex. This region contains a 46-nt U-rich element (68% U), and binding activity is effectively competed only by poly (U), unlabeled 3UTR2 RNA, and other U-rich RNA sequences, but not by poly(A), poly(G), and poly(C), or irrelevant RNA sequences which contain no U-rich element. Furthermore, cleavage of this segment within the U-rich region abolished the ability to form the complex. These data suggest that the U-rich element may be involved directly in RNA-protein interaction. Sequence data for this region of the 3`-UTR of human, rabbit, or rat SGLT1 are not available for comparison with the pig sequence in order to determine whether this URE motif is conserved across species.
Uridine-rich elements involved in destabilization of several nuclear transcription factor, cytokine, or lymphokine mRNAs have been divided into two broad categories (Chen and Shyu, 1994): those containing 1-3 copies of AUUUA motifs within the U-rich region, in some cases forming overlapping copies of the UUAUUUA(U/A)(U/A) nonamer, and those which lack the AUUUA motif. The SGLT1 URE falls within the latter category since it lacks an AUUUA motif within the U-rich element. Although three AUUUA motifs are present further downstream (Fig. 1), they do not participate in SG-URBP binding.
The functional significance of
SG-URBP binding to the U-rich mRNA sequence was not directly
demonstrated in the present study. SG-URBP binding activity was
activated by cyclic AMP-elevating agents and by phosphatase inhibitors.
The increase in half-life (8.6-fold) of SGLT1 3.9-kb mRNA after cyclic
AMP elevation (Peng and Lever, 1995) correlates well with the
5-10-fold increased formation of the SG-URBP complex under
parallel conditions (Table 1). Our previous results demonstrated
that inhibitors of protein synthesis did not block stabilization of the
message after cyclic AMP elevation (Peng and Lever, 1995). We propose
that the post-transcriptional stabilization of SGLT1 mRNA in response
to agents that elevate intracellular cAMP levels is mediated, at least
in part, by the interaction between SGLT1 mRNA and its specific binding
protein, SG-URBP. The ability of SG-URBP to bind its target site on the
mRNA would be activated by protein phosphorylation, directly or
indirectly mediated by protein kinase A. LLC-PK cells
express two distinct but similar protein kinase A catalytic subunits
encoded by different genes and both R
and R
regulatory subunits (Adavani et al., 1987). A protein
kinase A-deficient mutant of this cell line was deficient in
Na
/glucose cotransporter expression in addition to
alterations in other differentiated properties (Amsler et al.,
1991). The U-rich sequence (URE) identified in this study encompasses
the specific binding site for SG-URBP. This sequence may serve as a
component of the degradation machinery and participate in the selective
degradation of SGLT1 mRNA. It is downstream from the first
polyadenylation site suggesting it may function in the nucleus as well
as the cytoplasm. The URE may be a nuclease hypersensitive site or a
binding site for a destabilizing factor which directs nucleases to the
region. A potential hairpin loop region occurs upstream from the URE
but does not appear to participate in SG-URBP binding. Presumably, the
binding of phosphorylated SG-URBP would mask the U-rich sequence and
protect the message from degradation.
U-rich elements found within the 3`-UTRs of some short-lived mRNAs such as c-fos and c-myc play an important role in regulation of turnover of these mRNAs (Belasco and Brawerman, 1993; Chen and Shyu, 1995). Several U-rich element binding activities have been identified. These include a 59-kDa binding activity that interacts with a c-myc ARE in Balb/c3T3 cells (Alberta et al., 1994), a 37-kDa serum-inducible, translation-dependent protein that recognizes the c-fos ARE in NIH 3T3 cells (You et al., 1992), and a human elav-like neuronal protein 1 (Hel-N1) which specifically binds to a U-rich region in the 3`-UTR of c-myc mRNA (Levine et al., 1993) and forms a 28-kDa RNA-protein complex. We have observed a 28-kDa binding activity which also interacts with the URE of the SGLT1 3`-UTR. Whether this 28-kDa protein is a kidney cell counterpart of Hel-N1 or a different protein remains unknown. We observed that an increase in the 48-kDa SG-URBP binding activity after cyclic AMP elevation was consistently accompanied by a corresponding reduction in the formation of the 28-kDa complex. Both binding activities recognize the same 120-base region and are competed by U-rich RNAs. Therefore, these two RNA binding activities are reciprocally regulated by cyclic AMP and may share a common binding site.
Several AU-rich mRNA-binding protein activities can be modulated by reversible phosphorylation/dephosphorylation. The adenosine-uridine binding factor AUBF (Malter 1989) is a phosphoprotein which specifically binds to AUUUA motifs of several lymphokine, cytokine, and oncogene mRNAs (Gillis and Malter, 1991) after activation of protein kinase C by TPA and is proposed to contribute to agonist-induced stabilization of these labile mRNA species (Malter and Hong, 1991; Rajagopalan and Malter, 1994). Two cytoplasmic proteins of 65 and 45 kDa reported to stabilize prostaglandin endoperoxide synthase II (COX II) mRNA in response to IL-1b and TPA in renal mesangial cells specifically bind the 3`-UTR in a phosphorylation-dependent manner (Srivastava et al., 1994).
A group of AU-rich element (ARE)
mRNA-binding proteins in the M range of
32,000-40,000 (Brewer, 1991; Vakalopoulou et al., 1991;
Bohjanen et al., 1991, 1992; Bickel et al., 1992;
Port et al., 1992; Huang et al., 1993) bind to target
mRNAs containing both AUUUA pentameric motifs and flanking U-rich
domains. Within this category is a 35-kDa protein up-regulated by
isoproterenol which has been implicated in agonist-mediated
destabilization of
-adrenergic receptor mRNA (Huang et
al., 1993). However, AUUUA motifs are neither always necessary nor
always sufficient to target an mRNA for degradation (Shyu et
al., 1989; Kabnick and Housman, 1988; You et al., 1992;
Alberta et al., 1994). Comparative analysis has found an
obvious correlation between mRNA half-life and uridine content in the
immediate vicinity of the AUUUA motif (Alberta et al., 1994).
It appears that U-richness is the most prominent characteristic of
these AU elements. Binding of AUBF, a 44-kDa protein, to an artificial
80-bp reiterating AUUUA RNA transcribed from probe AU80, was stimulated
in extracts from cyclic AMP analog-treated adipocytes (Stephens et
al., 1992), but enhanced binding to the naturally occurring
message was not demonstrated. Our results suggest that AU80 and
c-fos ARE RNAs do not specifically interact with SG-URBP even
though both can effectively compete with the binding of SG-URBP to
SGLT1 3`-URE mRNA. Since SG-URBP differs from other known mRNA-binding
proteins not only in apparent molecular size, but also in activation by
agents that elevate intracellular cyclic AMP, it is probably a new
member of the U-rich region mRNA-binding protein inventory.
At present, we do not know if SG-URBP can also interact with a subset of mRNAs other than SGLT1 to coordinately influence their stability in response to cyclic AMP. Such a possibility would extend the Second Messenger Hypothesis for cyclic AMP-mediated regulation to the post-transcriptional level and involve RNA-binding phosphoproteins as components of this intracellular cascade mechanism.