Abstract
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 consrved. Taken together, these findings suggest a mechanism for modulation of NMDA receptor activity through the control of translational efficiency of a single subunit.
Highlights
Protein synthesis [4, 5]
Using the Xenopus oocyte expression system, it is shown that an encumbrance within the 5Ј-UTR1 of the NR2A subunit is capable of restricting its translation to 1% of the potential maximum
Expression of recombinant NMDA receptors in Xenopus oocytes has shown that the NR1 subunit can direct expression of functional homomeric channels [9]
Summary
Molecular Biology—Site-directed mutagenesis, in vitro transcription, and oocyte expression was performed as described previously [7]. Truncation mutations, mutations eliminating stem-loops, and the mutation of alternate start codon 3 utilized a single polymerase chain reaction. Mutations of alternate AUGs and rearrangement of stem 5 utilized the megaprimer polymerase chain reaction technique [8]. The sequence of the full-length ⑀1 cDNA 5Ј-UTR was determined by first subcloning a fragment containing the 5Ј-UTR into the M13 vector allowing for single-stranded DNA sequencing. In vitro transcription was performed using T7 RNA polymerase and RNA cap structure analog, m7G(5Ј)ppp(5Ј)G (New England Biolabs) and other in vitro transcription reagents (Promega). All constructs were contained within a modified pBluescript vector (Stratagene) and shared the following vector-derived leader sequence: GAATTGGGCGGGCCCCCCCTCGAG. The full-length ⑀1 cDNA contained the following leader sequence: GAATTGGGCGGGCCCCCCCTCGAGGTCGACTCTAGAGGATCC. All cDNAs were linearized with NotI (New England Biolabs) before run-off transcription.
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