(Received for publication, December 4, 1995; and in revised form, January 17, 1996)
From the
The N-methyl-D-aspartate (NMDA) receptor plays a central role in such phenomena as long term potentiation and excitotoxicity. This importance in defining both function and viability suggests that neurons must carefully control their expression of NMDA receptors. Whereas the NR1 subunit of the NMDA receptor is ubiquitously transcribed throughout the brain, transcription of NR2 subunits is spatially and temporally controlled. Since heteromeric assembly of both subunits is required for efficient functional expression, post-transcriptional modification of either subunit would affect NMDA receptor activity. Here it is demonstrated that the 5`-untranslated region (5`-UTR) of the NR2A subunit severely restricts its protein translation in both Xenopus oocytes and in an in vitro translation system. Mutational analysis of the 5`-UTR implicates secondary structure as the major translational impediment, while the five alternate start codons play minor roles. An important biological role for the 5`-UTR of NR2A is further suggested by the unusually high level of sequence conservation between species. In contrast, the 5`-UTR of NR1 does not inhibit translation and is not conserved. Taken together, these findings suggest a mechanism for modulation of NMDA receptor activity through the control of translational efficiency of a single subunit.
Fast synaptic excitatory neurotransmission within the central nervous system is mediated primarily by the neurotransmitter glutamate (1) . Of the class of ligand gated ion channels responsive to glutamate, the NMDA receptor possesses a unique calcium permeability. This property, along with the voltage-dependent magnesium block, governs the role of the NMDA receptor in such phenomena as long term potentiation and excitotoxicity(1) . NMDA receptor function is also critical for proper organization of synaptic networks during development(2) . The importance of modulation of NMDA receptor activity is exemplified in models of excitotoxicity, in which unrestricted channel activity induces neurodegeneration through excessive calcium influx(3) . Thus, a delicate balance between essential neuronal function and survival suggests that the quantity of functional NMDA receptors must be controlled. The initial cellular mechanism for control of protein expression resides at the level of transcription. However, translational regulation has also been proposed as a means of directing protein synthesis(4, 5) .
The NR1 subunit of the NMDA receptor is ubiquitously transcribed
throughout the brain. In contrast, NR2 subunits display distinct
regional and developmental transcription profiles. Xenopus laevis oocytes injected with NR1 mRNA form functional channels with small
agonist responses. Although NR2 subunits do not form functional
receptors when expressed alone, they enhance function when coinjected
with NR1. The importance of NR2 subunits is emphasized by the
behavioral deficits that result from the targeted disruption of the
NR2A gene(6) . Here, using the Xenopus oocyte
expression system, it is shown that an encumbrance within the 5`-UTR ()of the NR2A subunit is capable of restricting its
translation to 1% of the potential maximum. Thus, it is demonstrated
that the 5`-UTR of a single subunit is able to control NMDA receptor
activity over a range of three orders of magnitude. Furthermore, it is
recognized that the 5`-UTR of the NR2A subunit is highly conserved
across species. These findings suggest a conserved mechanism for
translational control of NMDA receptor activity.
Figure 2:
SDS-PAGE of in vitro translation
products. A rabbit reticulocyte lysate system was used to translate the
cRNAs indicated above the lanes (nomenclature is the same as in Fig. 1). Full-length NR1 and 1 containing the short 5`-UTR
of drk1 translated efficiently, yielding major protein bands that ran
close to their predicted molecular weights. Full-length
1
translated very poorly. A faint band of the proper molecular weight was
only seen after overloading the gel and overexposing the film (data not
shown).
Figure 1:
The 5`-UTR of 1 (mouse NR2A)
hinders translation in oocytes. Representative current traces obtained
using two-electrode voltage clamp recordings of oocytes injected with
equal quantities of cRNAs encoding NR1, NR1+NR2A (
1 with
full-length 5`-UTR), or NR1+NR2A-drk1 5`-UTR (
1 containing
5`-UTR of drk1). Coapplications of 100 µML-glutamate and 10 µM glycine are
represented by shaded bars. Cells were maintained at a holding
potential of -50 mV. Note that scale bars represent 5 s and
either 10 nanoamperes (A) or 10 microamperes (B).
Expression of recombinant NMDA receptors in Xenopus oocytes has shown that the NR1 subunit can direct expression of
functional homomeric channels(9) . NR2 cDNA clones do not form
functional channels unless coexpressed with NR1(11) . Our
initial attempts to express heteromeric NMDA receptors in Xenopus
laevis oocytes failed to produce robust agonist responses (Fig. 1). In an effort to increase the level of expression of
the NR1 + 1 (murine NR2A) combination, the 5`-UTR of
1
was replaced with that of drk1, a K
channel cDNA clone
known to translate well in Xenopus oocytes(12) . The
resulting agonist responses were dramatically increased over those
using the
1 cDNA containing the full-length 5`-UTR (Fig. 1). A similar replacement of the NR1 5`-UTR (265
nucleotides in length) with that of drk1 (13 nucleotides) produced no
change in the agonist response of homomeric channels (n = 20 oocytes, p > 0.4).
The inhibitory role of the NR2A 5`-UTR on translation could be a unique characteristic of the Xenopus oocyte expression system. We therefore studied the effect of the 5`-UTR on translation efficiency in a mammalian in vitro expression system. Full-length NR2A cRNA translated very poorly in a reticulocyte lysate system, while replacement of the NR2A 5`-UTR with the 5`-UTR of drk1 dramatically enhanced translation (Fig. 2). Thus the inhibitory effect of the NR2A 5`-UTR is consistent in both amphibian oocytes and a mammalian cell-free system.
Since the 1 cDNA nucleotide sequence present in GenBank(TM)
(accession number D10217) did not contain information on the 5`-UTR (10) , DNA sequencing of this region (282 nucleotides) was
performed (Fig. 3). This revealed that more than 99% identity
exists between the 5`-UTRs of the mouse
1 clone and the published
rat NR2A sequence(11) . Table 1, which compares
conservation of coding regions and 5`-UTRs for NR1 and NR2A in three
species(9, 13, 14, 15) , illustrates
the extraordinary degree of conservation among the NR2A 5`-UTRs.
Figure 3:
Graphical representation of the 5`-UTR of
1 (mouse NR2A). The beginning of the coding sequence for the
full-length ORF is shown in boldface. Alternate AUGs are
outlined and numbered according to their appearance in the linear
sequence. Locations of the 5`-most nucleotide of each of the serial
truncation mutations is indicated by the boxed numbers. The
5`-most nucleotide of the rat NR2A cDNA is symbolized by a boxed r and that of the human NR2A cDNA by a boxed h. The two boxed nucleotides mark the positions where the rat sequence is
found to diverge from that of the mouse sequence, the seven circled nucleotides mark positions of divergence of the human sequence
from that of the mouse sequence. The nucleotides outlined and labeled
as STEM 4 were replaced with the sequence 5`-TCT-3`, those of
the shaded and labeled STEM 5 with 5`-CGCGCC-3`. The
LOOP label outlines the nucleotides deleted in the ``loop''
mutation of stem-loop 5. The hairline under nucleotides
-65 through -21 illustrates the sequence exhibiting
homology with the 5`-UTR of G
. Self-complementary
structures were predicted by the StemLoop program in the Wisconsin
Package of the Genetics Computer Group (Madison,
WI).
The large boost in expression level caused by replacement of the long 5`-UTR of NR2A with a 13 nucleotide sequence inferred the presence of negative regulatory elements within the 282 nucleotides. The scanning hypothesis for initiation of protein translation predicts that elements upstream of the start codon for the full-length open reading frame (ORF) are responsible for inhibiting translation(5, 16) . In the 5`-UTR region of the NR2A cDNA, these include five alternative start codons (AUGs) giving rise to small open reading frames (µORFs), as well as possible structural impediments to the ribosome (i.e. self-complementary regions). Serial truncations of the NR2A 5`-UTR were generated and expressed in Xenopus oocytes in order to identify regions responsible for the inefficient translation (Fig. 4). There was no significant difference between the expression level of the full-length NR2A (282 nucleotides) and the mutation truncated at nucleotide number -196. This suggested that the primary inhibitory elements existed within the final 196 nucleotides of the NR2A 5`-UTR. Maximum expression was achieved by truncation at nucleotide number -68, suggesting that these inhibitory elements lie within the 128 nucleotides intervening nucleotides -196 and -68.
Figure 4:
Effect
of serial truncation of the 5`-UTR of NR2A (1).
Electrophysiological measurements as described in Fig. 1were
performed on oocytes injected with combinations of NR1 and each of the
indicated NR2A mutants. The chart represents subsequent comparisons of
the resulting measurements with measurements of oocytes injected with
the NR1+
1 combination containing the full-length
1
5`-UTR. The inset shows the incremental boosts conferred by
successive truncations as compared with the previous (longer)
construct. Error bars represent standard errors of the mean
(S.E.). p values of less than 0.001 are denoted by**, and p < 0.05 by *. N.S. indicates no significant
change from the NR1+
1 combination. Measurements were
performed on at least four batches of oocytes for each construct (11
batches total) and at least five oocytes for each batch. Mean current
measurements and total oocytes measured are as follows: NR1 alone, 14
± 11 nA, n = 56; NR1+
1, 143 ±
50 nA, n = 27; NR1+
1-196, 99 ±
20 nA, n = 40; NR1+
1-157, 1.0 ±
2.4 µA, n = 45; NR1+
1-114, 10.3
± 1.6 µA, n = 25; NR1+
1-68,
15.9 ± 1.7 nA, n = 20; NR1+
1-13,
15.6 ± 1.4 µA, n = 21; NR1+
1-drk1
5`-UTR, 9.8 ± 2.0 µA, n =
20.
In order to investigate the importance of alternate start codons for the incremental boosts found between the serial truncations, the 5`-most AUG was mutated in each of the parent constructs that preceded a boost (Fig. 5). Mutation of AUG 3 and AUG 5 (see Fig. 3) were each found to exhibit an approximate 2-fold increase in the efficiency of translation over those of their respective parent constructs, -196 and -114. To investigate the importance of context, we also generated the AUG 5 mutation within the base construct, -196. This again resulted in an approximate 2-fold increase in translational efficiency. These results suggest that less than 5% of the translational inhibition can be attributed to the presence of alternate start codons within the 5`-UTR of NR2A and that their inhibitory effects are not affected by preceding nucleotides. Finally, the small boost in translational efficiency occurring between truncation mutants -114 an -68 can be explained by the presence of AUG 5.
Figure 5:
Effect
of mutations of individual alternate AUGs. Measurements of whole
current responses are charted for each of the parent truncation mutants
and their respective AUG mutations. Error bars represent S.E.
values. p values of less than 0.001 are denoted by** and p < 0.05 by *. The inset shows the comparison of AUG
mutations normalized to parent constructs. Measurements were performed
on at least three batches of oocytes for each construct and at least
five oocytes for each batch. Mean current measurements and total
oocytes measured are as follows: NR1+1-196
AUG3,
191 ± 33 nA, n = 20;
NR1+
1-196
AUG5, 181 ± 35 nA, n = 15; NR1+
1-157
AUG4, 1.5 ± 0.5
µA, n = 25; NR1+
1-114
AUG5,
18.8 ± 1.0 µA, n =
20.
The minimal effects conferred by alternate start codons suggested an important inhibitory role for secondary structures within the NR2A 5`-UTR. Although the precise structure of the NR2A mRNA is not known, several algorithms exist which can predict possible stem-loop structures based on self-complementary regions within the 5`-UTR. One such algorithm was employed in the generation of the putative structure shown in Fig. 3. The results obtained thus far suggested that most of the translational inhibition can be localized to the nucleotides between -196 and -114. Thus, the contribution to inhibition of translational efficiency of the two putative stem-loop structures existing within this region were examined.
Removal of the nucleotides comprising the predicted loop structure of stem-loop 5 results in a 10-fold decrease in translation efficiency versus that of the parent construct, -157 (Fig. 6). This diminution suggests that the loop structure plays a role in relieving translational inhibition, possibly by decreasing the stability of stem 5. We then examined the role of stem-loop 5 within the context of the base construct -196. A stretch of six nucleotides within the stem was mutated such that the overall G/C content would not be diminished, yet the predicted loop structure would be expanded. No effect on translational efficiency is evident from this mutation. The final mutation of secondary structure targeted stem-loop 4. To avoid consequently placing AUG 3 into an unusual context, we generated a mutant lacking both stem-loop 4 and AUG 3. This resulted in a 40-fold increase in translation efficiency (Fig. 6).
Figure 6:
Mutations affecting putative secondary
structures. Measurements of whole current responses are charted as in Fig. 4. The inset shows the relative decrease from the
deletion of the loop structure in stem-loop 5 and the relative increase
from the deletion of stem-loop 4. Error bars represent S.E.
values. p values of less than 0.001 are denoted by**.
Measurements were performed on three batches of oocytes for each
construct and five oocytes for each batch. Mean current measurements
and total oocytes measured are as follows:
NR1+1-157
LOOP, 84 ± 9 nA, n = 15; NR1+
1-196
STEM5, 114 ± 20
nA, n = 15;
NR1+
1-196
AUG3
STEM-LOOP4, 114 ± 20 nA, n = 15.
Finally, it has been demonstrated that certain mRNAs can be
translated in a cap-independent fashion(17) . Therefore, the
synthetic mG cap analog was omitted from an in vitro translation preparation of full-length NR2A. This resulted in a
predicted reduction in the translational efficiency. Uncapped,
full-length NR2A did not result in a detectable boost of expression
level when coinjected with NR1 (n = 21, p >
0.3). Thus, translation of full-length NR2A is cap-dependent.
Considerable data have been generated documenting the importance of 5`-UTRs in defining the translatability of mRNAs(16, 17, 18) . The longstanding ribosome scanning model has adequately predicted many of the findings, although several alternative models have arisen to explain specific aberrant cases of translational control(5, 17, 19, 20) . Furthermore, of the relatively few mRNAs which posses 5` encumbrances, most fall into several categories of proteins critically important to maintaining cellular viability (e.g. growth hormone receptors and proto-oncogenes)(5) . A leading hypothesis suggests that translation of mRNAs containing structured 5`-UTRs might be selectively modulated through the regulation of helicases capable of relieving structural impediments(17, 20) . Indeed, evidence of this mechanism involving control of specific translation initiation factors supports this hypothesis. Modulation may occur through the phosphorylation and consequent disruption of the binding properties of a protein (4E-BP1) which otherwise inhibits a critical initiation factor (eIF-4E)(4) . Alternatively, the presence of extensive structural configurations may also decrease the global rate of translation through a mechanism involving a double-stranded RNA-activated protein kinase (PKR)(20) . Finally, instances of translational control defined by developmental stage and tissue specificity have also been documented(18, 21) .
The NR1 subunit is essential for expression of functional NMDA receptors(9) . However, electrophysiological measurements demonstrate that homomeric NR1 channels express quite poorly in Xenopus oocytes and that apparently these channels are functionally undetectable in mammalian heterologous expression systems. Indeed, here we show that for Xenopus oocytes, functional activity of homomeric NR1 receptors is only 0.1% of that of heteromeric receptors utilizing efficiently translating NR2A subunits (Fig. 1). Therefore, efficient functional expression requires both NR1 and NR2 subunits.
In vitro translation of the
full-length 1 cRNA using a rabbit reticulocyte lysate system
mimicked the results found in Xenopus oocytes (Fig. 2).
Apparent molecular weights predicted to arise from initiation at
alternate AUGs 1 through 5 are 3.5, 11.9, 2.1, 1.1, and 7.0
kilodaltons, respectively. The spurious band seen on the SDS-PAGE gel
at approximately 75 kilodaltons may represent either premature
termination or degradation of the
1 protein. Thus, no peptide
products from the alternate µORFs were evident. This finding is
consistent with previous reports suggesting that these AUGs may only
temporarily ``stall'' the scanning ribosome (5) . The
consistency of findings between the two systems argues that the
analysis performed on translational inhibition in oocytes is
representative of the inhibitory mechanisms of mammalian cells.
Furthermore, because this analysis involves RNA that is transcribed in vitro and microinjected into the oocyte cytoplasm, the
inhibition would not be expected to involve the oocyte mRNA masking
phenomenon(22) .
No cell-specific transcriptional control over the NR1 gene is apparent beyond that of alternative splicing and restriction to the central nervous system(23) . In contrast, the NR2 subunits are regulated according to developmental stages and cell types(24, 25) . While functional NMDA receptors require the constitutively expressed NR1 subunit, the results presented here infer that the biological relevance of these receptors is regulated by the controlled expression of the NR2 subunits. Furthermore, our results suggest that some of this regulation may be imparted by control of translational efficiency.
This study demonstrates that the translational control mechanism is present only in the 5`-UTR of the NR2 subunit and not in the 5`-UTR of the NR1 subunit. The difference in translatability of the two messages does not arise from any general features of the 5`-UTRs. The 5`-UTRs of both subunits possess similar nucleotide lengths, identical overall G/C content and comparable GC profiles (Fig. 7). Emphasis of the contrast is rendered by the sequence homology comparisons shown in Table 1. Homology comparisons of the coding sequences demonstrate that both subunits exhibit a similar degree of conservation (approximately 95% versus rat and 90% versus human). However, within the 5`-UTRs of the two subunits, relatively little conservation is seen for the NR1 subunit in comparison with the very high degree of conservation for the NR2 subunit. The relative level of sequence homology alone argues for a pertinent role for the 5`-UTR of the NR2A subunit.
Figure 7:
Profiles of GC content for 5`-UTRs of
1 and NR1. Local GC content of the 5`-UTR was estimated as
described under ``Experimental Procedures'' for both
1
and NR1. The positions of the different truncations as well as putative
stem-loops 4 and 5 are indicated. GC content per se does not seem to
explain the difference in translatability between the two NMDA receptor
subunits. The overall GC content of the 5`-UTR is 67% for both NR1 and
1. Also, NR1 has a local maximum which is higher and more
extensive than the maximum at stem-loop 4 in
1.
The gene sequence for the murine NR2C subunit has been determined(26) . The fact that this NR2 subunit is alternatively spliced only within its 772 nucleotide long 5`-UTR again is suggestive of a role in translational regulation. No gene sequence is available for the NR2A subunit. However, a Northern blot of the NR2A subunit displays a major band of approximately 18 kilobases(10) . If this huge message represents unspliced pre-mRNA, then this would suggest the application of another post-transcriptional regulatory mechanism (i.e. via controlled splicing). This has been previously reported for other messages(5) . We cannot rule out the possibility that a significant portion of the endogenous 5`-UTR remains unrepresented in the cDNA clone. The addition of upstream nucleotides may relieve impediments found within the 5`-UTR of the NR2A cRNA, although our results provide no support for this hypothesis.
Our results do suggest that control of translational efficiency is largely derived from the 128 nucleotides found between nucleotide numbers -196 and -68. Little of the inhibition appears to arise from the five alternate start codons found within the 5`-UTR. Table 2compares the sequences surrounding the coding and alternate AUGs with the Kozak consensus sequence. The alternate AUGs show a less favorable context than the coding AUGs. In particular, they are missing the important G at position +4. The surrounding nucleotide sequence is important for the AUG of the full-length ORF. A near-perfect sequence is present in the mutant truncated at nucleotide number -13. This truncation mutation translates more efficiently than the construct containing the 5`-UTR of drk1 (p < 0.001, Fig. 4), which possess identical length and less consensus integrity.
The relatively small impact of the alternate start codons implies that the dominant effect is due to secondary structure. A structural model of the 5`-UTR was generated in order to facilitate the planning of experiments testing the relative effects of secondary structure on translational inhibition. During the construction of this model, several interesting regions of sequence homology were recognized. Of minor notation is the observation that 10 of 12 nucleotides within the loop of proposed stem-loop structure 5 display homology with the loop of the trans-acting responsive (TAR) element structure of human immunodeficiency virus-1(27) . Although loop 5 possesses 70% G/C content, its deletion identified it as a positive regulator of translational efficiency. However, rearranging several nucleotides found within the stem portion of stem-loop 5 in an attempt to expand this putative loop structure did not provide further relief of translational inhibition. This result may be due to a rematching of the scrambled nucleotides into a new stem-loop configuration.
In contrast, the elimination of stem-loop structure 4 (93% G/C content) provided considerable relief of inhibition, clearly substantiating its role as a negative regulator of translation. The relief provided by the elimination of stem-loop 4 was greater than what would be expected based on the results of the truncation mutations. This is likely due to the induction of a novel folding structure of the mRNA. Alternatively, the elimination of stem-loop 4 may have added an increment of unmatched nucleotides between the 5`-cap and downstream self-complementary structures. This addition may have permitted ample initiation access for the ribosome to unwind downstream secondary structures of lesser stability as has been reported previously(5) . Another alternative explanation for these findings which has not yet been considered is the possible role of mRNA-binding proteins. It can be argued that specific sequences within the 5`-UTR may be capable of recruiting endogenous molecules which can act to stimulate or inhibit translation(18) . This study provides no evidence to either substantiate or refute this hypothesis.
A more significant region of
homology was recognized between the 5`-UTRs of NR2A and the
-subunit of the heterotrimeric G-protein, G
. These 38
nucleotides are 76% identical and include an uninterrupted stretch of
15 shared nucleotides. Evidence of translational regulation of
G
has also been reported, as well as a high degree of
sequence conservation within the 5`-UTRs across species(28) .
Finally, the intramolecular location of these homologous regions are
downstream of the regions identified as inhibitory to translation of
both mRNAs, hinting that these sequences may play similar roles in both
messages.
Although there is no in vivo study correlating quantities of mRNA with protein levels of NMDA receptor subunits, several observations are suggestive of post-transcriptional control mechanisms. In a temporal study utilizing primary cultures of cortical neurons, an increase in NMDA receptor density as measured by antagonist binding is not accompanied by a change in mRNA levels(29) . Similarly, the observation of an NMDA receptor subtype-specific increase in binding density in response to kindling of adult male rats does not arise from quantitative alterations in subunit transcripts(30) . Although increases in binding density can be explained by other post-transcriptional alterations, these dichotomies of apparent protein and mRNA levels could emanate from a selective translational control mechanism.
mRNA, polyribosomes, and smooth endoplasmic reticulum (35) have been localized to dendritic processes(31, 32) . Therefore, it has been speculated that synaptic activity may influence translational efficiency(33) . Because the cellular organelles necessary for the assembly of integral membrane proteins (i.e. rough endoplasmic reticulum and Golgi apparatus) have not been observed at post-synaptic densities, this model has been restricted to include only cytoplasmic proteins. However, it is interesting to note that glutamate receptor subunit mRNAs have also been identified in neuronal processes(34) .
This study has recognized that the 5`-UTR of the NMDA receptor NR2A subunit is capable of modulating the activity of this critical protein complex over 3 orders of magnitude. Little of the inhibition conferred by the 5`-UTR involves its five alternate AUGs. A large translational impediment is localized to a G/C-rich stretch of 15 nucleotides. The presence of another stretch of 11 nucleotides is recognized as a positive modulator of translation. The extraordinary sequence conservation of these untranslated nucleotides argues for an endogenous role for this sequence. Finally, recent advances make it possible to surmise that this highly conserved, strongly inhibitive sequence may represent a neuronal mechanism for regulation of NMDA receptor activity through the manipulation of translational efficiency.