(Received for publication, November 14, 1995; and in revised form, February 21, 1996)
From the
HuD is a human neuronal specific RNA-binding protein. In this study we have purified HuD and examined its RNA binding properties in detail. HuD binds to mRNAs that contain an AU-rich element with high affinity. In the case of the c-fos AU-rich element, HuD binds to a 27-nucleotide core element comprising AUUUA, AUUUUA, and AUUUUUA motifs. Mutation in any two of these motifs abrogates binding. HuD contains two tandem RNA recognition motifs (RRM), a basic domain, and a third RRM. Deletion analysis has shown that only the first and second RRMs are essential for RNA binding. Thus, these specific RNA binding properties support the idea that the HuD regulates gene expression at the post-transcriptional level.
HuD is a human member of the Elav-like neuronal RNA-binding protein family(1) . Elav, the founder member of this family, was discovered through genetic studies in Drosophila(2, 3) . Mutations in the Elav locus result in a dysfunctional nervous system in which neuroblasts continue to proliferate, fail to differentiate, and migrate inappropriately(4) . In addition to its role in development, Elav function is also continuously required for neuronal maintenance. Flies with temperature-sensitive mutations in Elav lose motor control on exposure to the restrictive temperature(5) . The human homologues of Elav were independently discovered as the target antigens in paraneoplastic sensory neuronopathy(1) . This observation suggests that vertebrate Elav-like proteins may also have a critical role in the development and maintenance of vertebrate neurons. Information on the Elav-like family has significantly expanded with the isolation of further homologues from human, mouse, rat and Xenopus(6, 7, 8, 9) . In Drosophila there appear to be only two genes, elav and rbp9, which differ in their spatial and temporal expression pattern. In higher organisms there appears to be an additional member (Hel-N1 in humans, elrB in Xenopus) that is distinct, in that it is expressed in testis and ovary and in neurons(8, 10) . In keeping with their likely role in neurogenesis the vertebrate Elav-like genes are expressed on terminal differentiation of neurons(6, 11, 12) .
All Elav-like
proteins contain three copies of the RNA recognition motif (RRM), ()an 80-amino acid domain found in many RNA-binding proteins
of diverse function(13, 14) . Thus, it is thought that
the Elav-like proteins promote neuronal differentiation by
post-transcriptional regulation of mRNAs that control cell
proliferation and differentiation. This hypothesis has been
strengthened by the observation that the Elav-like proteins selectively
bind to the 3`-UTR of mRNAs that regulate cell proliferation and
differentiation(10, 15) . It has been proposed that
the Elav-like proteins specifically bind to the short AU-rich segments
(AREs or ``Shaw-Kamen elements'') that regulate mRNA
turnover(16) . In particular, studies on Hel-N1 have shown that
it specifically selects mRNAs that contain AREs from a total mRNA
library(17) . Studies on HuD have shown that in crude extracts
it specifically binds to the ARE element of c-fos mRNA(18) . More detailed analysis of this interaction was
not possible, since highly purified proteins were not available.
Although many activities have been identified that bind to ARE elements
in crude extracts, relatively few have been purified and
cloned(19, 20, 21, 22) . Moreover,
after cloning and purification, such activities have not displayed the
same specificity displayed in the crude
extract(23, 24) . In this paper we examine the RNA
binding properties of the purified HuD protein and have established
that it binds to AREs with high affinity and specificity.
Figure 1: A, the structure of c-fos mRNA showing the location of the transcripts. B, the sequence of the AUFL transcript showing the HuD binding site (thick underline). The ARE is in large boldface lettering.
Figure 2:
HuD binds to the 3`-untranslated region of
c-fos mRNA. The indicated concentrations of GST or HuD were
mixed with P-labeled RNA (100 pM, 30,000 cpm/pmol
UTP). Following incubation at 37 °C for 10 min, 25% of the reaction
mixtures were resolved on 1% agarose gel.
Figure 3:
HuD binds to c-fos RNA but not to
globin RNA. P-labeled RNA (100 pM, 30,000
cpm/pmol UTP) was incubated with the indicated concentration of HuD
protein. After 10 min of incubation at 37 °C, 25% of the reaction
mixtures were resolved on 1% agarose gel.
Figure 4:
The effect of salt on RNA binding. P-labeled RNA (100 pM, 30,000 cpm/pmol UTP) was
incubated without protein or with HuD (240 nM) protein at the
indicated salt concentration. After 10 min of incubation at 37 °C,
25% of the reaction mixtures were resolved on 1% agarose
gel.
Figure 5:
A, the
time course of complex formation. P-labeled RNA (100
pM, 30,000 cpm/pmol UTP) was incubated with HuD (240
nM) protein. After incubating for the indicated time at 37
°C, 25% of the reaction mixtures were resolved on 1% agarose gel. Fig. 5B shows the graphical
representation.
Figure 6:
The affinity of HuD with c-fos and globin mRNAs as determined by nitrocellulose filtration. A, plot of percentage of RNA bound versus log of HuD
concentration. , c-fos;
, globin. B, plot
of log complex/free RNA versus log HuD concentration.
,
c-fos.
Figure 7:
A, HuD binds to the c-fos ARE. P-labeled RNA (100 pM, 30,000 cpm/pmol UTP) was
incubated without protein, with GST, or with the indicated
concentration of HuD protein. After 10 min of incubation at 37 °C,
25% of the reaction mixtures were resolved on 1% agarose gel. B, HuD affinity with AU1, AU2, and AU12 as determined by
nitrocellulose filtration.
, AU1;
, AU2; ┌, AU12;
, ARE;
, AUFL;
, globin.
Figure 8:
A, RNase T1 analysis of HuD-c-fos RNA complex. The indicated concentrations of HuD or GST were
incubated with P-labeled RNA (400 pM, 30,000
cpm/pmol UTP, lanes 3-7) at 37 °C for 10 min. After
treating the reaction mixture with RNase T1 (0.5 unit/reaction), the
reaction mixtures were filtered through nitrocellulose. The bound RNA
fragments were extracted and resolved on 12% denaturing polyacrylamide
gel. Lane 1, ØX174 HinfI fragments; lane
2, RNase T1 digestion of AUFL RNA. B, RNase T1-protected
fragments were eluted from the preparative gel and analyzed by
redigestion with RNase T1.
Figure 9:
RNase T1 analysis of HuD-c-fos RNA complex. The indicated concentrations of HuD or GST were
incubated with P-labeled RNA (400 pM, 30,000
cpm/pmol UTP, lanes 3-5) or AU12 RNA (400 pM,
30,000 cpm/pmol UTP, lanes 7-9) at 37 °C for 10 min.
After treating the reaction mixture with RNase T1 (0.5 unit/reaction),
the reaction mixtures were filtered through nitrocellulose. The bound
RNA fragments were extracted and resolved on 12% denaturing
polyacrylamide gel. Lane 1, ØX174 HinfI
fragments; lane 2, RNase T1 digestion of AUFL RNA; lane
6, RNase T1 digestion of AU12 RNA.
We next tested whether the 35-nucleotide segment could independently
support HuD binding. The 35-nucleotide segment and a series of
progressive 3` deletions called 27, 20, and 13 were chemically
synthesized (Fig. 10). The 35-nucleotide segment supported
complex formation with similar affinity (apparent K = 29 nM) as the AUFL transcript (Fig. 10).
Similar reactivity was also observed with the 27-nucleotide segment
(apparent K
= 28 nM), but deletion
of a further 7 nucleotides diminished reactivity (only 20% complex
formation at 720 nM). Deletion of a further 7 nucleotides
yielded no detectable complex formation even at 720 nM. Thus,
we concluded that the essential HuD recognition sequences reside in the
27-nucleotide segment. We also synthesized a 23-nucleotide fragment
(called 23) that encompasses the minor recognition site identified by
the T1 fragment A. As expected, this fragment was bound by HuD, albeit
with lower affinity (20% complex formation at 720 nM).
Figure 10:
Deletion analysis of the HuD binding
site. The indicated P end-labeled RNAs were incubated with
HuD and filtered through nitrocellulose. A, sequences of
deletions; B, RNA binding activity.
, 35;
, 27;
, 20;
, 13;
, 23.
We next examined the RNA binding specificity of HuD. Many RNA-binding proteins also recognize single-stranded DNA(14) . A DNA oligonucleotide corresponding to the sequences of the 27-nucleotide fos ARE was synthesized. As shown in Fig. 11, HuD binds exclusively to the RNA oligonucleotide but not to the DNA oligonucleotide.
Figure 11: HuD binds specifically to RNA.
Figure 12:
HuD
binding to c-fos ARE is displaced by the AdIVA ARE. Labeled AUFL RNA (100 pM, 30,000 cpm/pmol UTP) was
incubated with 240 nM recombinant HuD protein. The indicated
molar excess of IVA2 (CUGGUUUUUUAUUUAUGUUUUAAACC) or ``R''
(CUAGAGUUCAUCGCAAUUGCA) was added into the reaction. After 10 min of
incubation at 37 °C, 25% of the reaction mixtures were resolved on
1% agarose gel.
The most discernible structural feature of the
27-nucleotide segment is the arrangement of U residues. We therefore
tested the significance of these residues by synthesizing mutant
oligonucleotides in which they were changed to G and C residues. The
sequences of the mutant oligonucleotides are described in Fig. 13. Substitution of only one of the three U stretches had a
modest effect on binding (Fig. 13). The apparent K for the 27-nucleotide oligo was 11 nM,
whereas the apparent K
for mutants 27-2, 27-3, and
27-4 was 25, 38, and 62 nM, respectively. Disruption of any
two stretches (mutants 27-5, 27-6, 27-7) drastically reduced RNA
binding (only 5% at 720 nM HuD). Alteration of all three
stretches (mutant 27-8) eliminated binding (no complex formation at 720
nM HuD).
Figure 13:
Mutational analysis of the HuD binding
site. The indicated P-end-labeled RNAs were incubated with
HuD and filtered through nitrocellulose. A, sequences of
mutations; B, RNA binding activity. ┌, 27;
, 27-2;
, 27-3;
, 27-4;
, 27-5;
, 27-6;
, 27-7;
, 27-8.
To
determine the domains of HuD required for RNA binding we purified seven
mutant proteins that are shown in Fig. 14B. RNA binding
affinity was assayed by gel retardation, and protein-RNA complex
formation was quantitated by Cerenkov counting. We did not use the
nitrocellulose filter binding technique because one of the mutant
proteins (HuD I II) did not give similar results on gel retardation and
nitrocellulose filter binding assays. All other mutants displayed
similar K values on using either technique. The
apparent K
values for HuD, HuD I II B, and HuD I
II were 16, 98, and 125 nM, respectively. Thus, the third RNA
domain is not essential for RNA binding. As expected the third RNA
binding domain alone (HuD III) or in conjunction with the basic segment
(HuD BIII) displayed very low binding activity (only 5% activity at
7,200 nM HuD). Similarly the basic segment did not bind RNA,
nor did it significantly stimulate the activity of HuD I II. HuD I and
HuD II are required in tandem, since either alone displayed poor RNA
binding (apparent K
values of 2,100 and 2,000,
respectively).
Figure 14:
A, analysis of RNA binding domains. The
indicated concentrations of purified HuD derivatives were mixed with P-labeled AUFL RNA (100 pM, 30,000 cpm/pmol UTP).
Following incubation at 37 °C for 10 min, 25% of the reaction
mixtures were resolved on 1% agarose gel. B, structure of the
mutant HuD derivatives. The residues of HuD contained in each construct
are as follows: for pGEX-HuD I II B, 2-268; for pGEX-HuD I II,
2-216; for pGEX-HuD I, 28-136; for pGEX-HuD II,
110-216; for pGEX-HuD B, 201-297; for pGEX-HuD B III,
245-373; for pGEX-HuD III, 279-373. C,
quantitative analysis of mutant binding activity.
, HuD;
,
I II B;
, I II;
, I;
, II;
, B;
, B III;
&cjs2114;, III.
These results provide new and more extensive information on the RNA binding properties of HuD, a member of the Elav-like family(8) . We have shown that HuD specifically binds to the c-fos ARE. Such ARE elements are conspicuously present in the 3`-UTR of mRNAs that are rapidly and transiently expressed after cellular stimulation (16) . Moreover, it is clear that the rapid induction of such mRNAs is the result of an inhibition of ARE-mediated degradation. Thus, the present studies further strengthen the hypothesis that the Elav-like proteins (HuD, HuC, and Hel-N1) control neuronal maintenance and development by the post-transcriptional regulation of gene expression (10, 18) . Such rapid and transient expression of genes is of peculiar importance to neuronal cells(37) . Neuronal cells are continuously stimulated by neurotransmitters with consequent transient alterations in gene expression. It is not surprising, therefore, that the Elav family has diverged in higher organisms and encompasses three family members. It is important to note that this form of post-transcriptional control occurs in all cell types. It is likely that a homologue of the Elav-like proteins will be expressed in all cells. Recently such an activity has been cloned and characterized(8, 38) .
We have focused on the
interaction between HuD and c-fos mRNA because its ARE has
been well characterized. When the c-fos ARE was inserted into
the 3`-UTR of -globin mRNA, it decreased the half-life of this
stable message from 24 h to 37 min(32) . We have shown that HuD
binds to the ARE element but not to the adjacent AU-rich elements AU1
and AU2. Although AU1 and AU2 are well conserved in chicken, rat, and
human fos mRNA, they appear to contribute little destabilizing
activity(32) . Using T1 digestion and deleted transcripts we
have determined the minimal sequences recognized by HuD. The salient
structural feature of the c-fos ARE, as in all AREs, is the
presence and organization of AUUUA pentanucleotide
sequences(16, 39) . The low affinity HuD binding site
comprises a tandem AUUUA motif. The major binding site, which exhibits
a similar affinity as the entire transcript, is comprised of an AUUUA
element, an AUUUUA hexamer, and an AUUUUUA heptamer (Fig. 8).
The low affinity of the tandem AUUUA and mutation of the third AUUUA
does not significantly affect binding, indicating that the AUUUA motifs
themselves are not essential for binding. Disruption of the AUUUUUA
heptamer, however, in addition to mutation of the AUUUA or AUUUUA
motifs abrogates binding. From these results we conclude that HuD does
not recognize a primary sequence but binds to a structure that requires
the participation of at least two of the three U stretches. This idea
fits with our observation that HuD binds to and is displaced by other
AREs of disparate sequence but presumably similar structure.
Several ARE binding activities have been previously described(19, 20, 21, 22, 40, 41, 42) . The majority of these studies have utilized a UV cross-linking assay. Thus it is difficult to make a comparison with our results. One of these activities (AUF1) has been purified and cloned and is clearly a different gene product(24) . In contrast to the tripartite structure of HuD, AUF1 contains two RRMs and a C-terminal glutamine-rich domain. The RNA binding properties shown here most resemble the activity identified by Vakalopoulou et al. The 32-kDa binding protein described by these authors required both the AUUUA and surrounding U-rich flanking sequences for maximal binding(21) . More informed comparisons between the previously described RNA binding activities and HuD must await their purification and cloning.
HuD contains an N-terminal domain followed by two
tandem RRM domains, a basic domain, and a third RRM. RRMs are
operationally defined by an 80-90-amino acid segment containing
two highly conserved motifs called RNP1 and
RNP2(43, 44, 45) . Most RNA-binding proteins
have a modular arrangement of such RRMs interspersed with various
auxiliary segments. In some cases, the auxiliary segment also binds RNA (29) or in others contributes to the RNA binding specificity of
the RRM. We have found that only the first and second tandem RRMs of
HuD are required for binding to AREs. The auxiliary basic segment does
not bind to RNA, nor does it stimulate the activity of the RRMs. Thus,
it is likely that it is involved in protein-protein interactions. It is
also possible that it may bind to other RNA sequences. Since the third
RNA binding domain only binds RNA with very low affinity, it is
feasible that it binds to a different RNA target. This target could be
another mRNA or a small RNA involved in mRNA degradation. Recently we
have observed that it binds to the poly(A) tail of mRNA. ()These results are dissimilar to similar studies performed
on Hel-N1. Levine et al.(15) observed that the third
RRM was essential for ARE binding. This result is surprising in view of
the fact that the individual RRMs are very well conserved between
Elav-like family members.
Thus, the data presented here points to a role of HuD in the regulation of mRNA turnover. At this point we can only speculate on the precise role. The purified protein does not exhibit any detectable endonuclease activity with ARE- or non-ARE-containing RNAs (data not shown). Thus, we believe that HuD binds to the ARE and recruits a larger complex that can degrade mRNA. Given that HuD binds to the 3` end of the c-fos ARE, we think that it may play a role in deadenylation, the first step in selective mRNA degradation(30) . If this model is correct, purified HuD may provide a reagent to assay and characterize such an activity and thereby reconstitute the first steps in selective degradation.