( $\alpha$ -amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid) receptor is an important component of the human nervous system. It is present in several forms, in different neurons, and some of this variety results from posttranscriptional modification. This problem explores research on the mechanism of this RNA editing.
An initial report by Sommer and coauthors (1991) looked at the sequence encoding a key Arg residue in the AMPA receptor. The sequence of the cDNA (see Fig. 9-14) for the AMPA receptor showed a CGG (Arg; see Fig. $27-7$ ) codon for this amino acid. Surprisingly, the genomic DNA showed a CAG (Gln) codon at this position.
(a) Explain how this result is consistent with posttranscriptional modification of the AMPA receptor mRNA.
Rueter and colleagues (1995) explored this mechanism in detail. They first developed an assay to differentiate between edited and unedited transcripts, based on the Sanger method of DNA sequencing (see Fig. $8-34$ ). They modificd the technique to determine whether the base in question was an A (as in CAG) or not. They designed two DNA primers based on the genomic DNA sequence of this region of the AMPA gene. These primers, and the genomic DNA sequence of the nontemplate strand for the relevant region of the AMPA receptor gene, are shown at the bottom of the page; the A residue that is edited is in red.
To detect whether this A was present or had been edited to another base, Rueter and coworkers used the following procedure:
1. Prepared cDNA complementary to the mRNA, using primer 1, reverse transcriptase, dATP, dGTP, dCTP, and dTTP.
2. Removed the mRNA.
3. Annealed 32 P-labeled primer 2 to the cDNA and reacted this with DNA polymerase, dGTP, dCTP, dTTP, and ddATP (dideoxy ATP; see Fig. $8-34$ ).
4. Denatured the resulting duplexes and separated them with polyacrylamide gel electrophoresis (see Fig. $3-18$ ).
5. Detected the $^{32}$ P-labeled DNA species with autoradiography.
They found that edited mRNA produced a 22 nucleotide $\left[^{32} \mathrm{P}\right] \mathrm{DNA},$ whereas unedited mRNA produced a 19 nucleotide $\left[^{32} \mathrm{P}\right]$ DNA.
(b) Using the sequences below, explain how the edited and unedited mRNAs resulted in these different products.
Using the same procedure, this time to measure the fraction of transcripts edited under different conditions, the researchers found that extracts of cultured cpithelial cells (a common cell line called HeLa) could edit the mRNA at a high level. To determine the nature of the editing machinery, they pretreated an active HeLa cell extract as described in the following table and measured its ability to edit AMPA mRNA. Proteinase K degrades only proteins; micrococcal nuclease, only DNA.
(c) Use these data to argue that the editing machinery consists of protein. What is a key weakness in this argument?
To determine the exact nature of the edited base, Rueter and colleagues used the following procedure:
1. Produced mRNA, using $[a-32]$ ATP in the reaction mixture.
2. Edited the labeled mRNA by incubating with HeLa extract.
3. Hydrolyzed the edited mRNA with nuclease P1 to produce single nucleotide monophosphates.
4. Separated the nucleotide monophosphates with thin-layer chromatography (TLC; sce Fig. $10-25 \mathrm{b}$ ).
5. Identified the resulting 32 p-labeled nucleotide monophosphates with autoradiography.
In unedited mRNA, they found only $\left[^{32} \mathrm{P}\right] \mathrm{AMP} ;$ in edited mRNA, they found mostly $\left[^{32} \mathrm{P}\right] \mathrm{AMP}$ with some $\left[^{32} \mathrm{P}\right] \mathrm{IMP}$ (inosine monophosphate; see Fig. $22-36$ ).
(d) Why was it necessary to use $\left[\alpha-^{32} \mathrm{P}\right] \mathrm{ATP}$ rather than $\left[\beta^{32} \mathrm{P}\right] \mathrm{ATP}$ or $\left[\gamma^{32} \mathrm{P}\right] \mathrm{ATP}$ inthis experiment? (e) Why was it necessary to use $\left[a^{32} \mathrm{P}\right] \mathrm{ATP}$ rather than $\left[a-^{32} \mathrm{P}\right] \mathrm{GTP},\left[\alpha-^{32} \mathrm{P}\right] \mathrm{CTP},$ or
$\left[a-^{32} \mathrm{P}\right] \mathrm{UTP} ?$
(f) How does the result exclude the possibility that the entire A nucleotide (sugar, base, and phosphate) was removed and replaced by an I nucleotide during the editing process? The researchers next edited mRNA that was labeled with $\left[2,8-^{3} \mathrm{H}\right] \mathrm{ATP}$ and repeated the above procedure. The only $^{3}$ H-labeled mononucleotides produced were AMP and IMP.
(g) How does this result exclude removal of the A base (leaving the sugar-phosphate backbone intact) followed by replacement with an I base as a mechanism of editing? What, then, is the most likely mechanism of editing in this case?
(h) How does changing an A to an I residue in the mRNA explain the Gln to Arg change in protein sequence in the two forms of AMPA receptor protein? (Hint: See Fig. 27-8.)
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