Neuronal Expression of Neural Nitric Oxide Synthase (nNOS) Protein Is Suppressed by an Antisense RNA Transcribed from an NOS Pseudogene

Alignment of the antisense region (93–238 nt) of the pseudo-NOS transcript with its complementary counterpart in the nNOS mRNA. In this alignment, there is ∼80% complementarity. The non-Watson–Crick G–U base pairs that are common in RNA secondary structure are shown by dots. The positions of three primers used in the identification of RNA–RNA duplexes areunderlined and named. Primers in positions expected to be protected from ribonuclease A (within the proposed duplex) are P2 and P3. The primer located outside of the protected area is called P1. Further explanation of the ribonuclease A protection experiment is provided in Figure 5. Details of procedures are in Materials and Methods.

The pseudo-NOS and nNOS-encoding transcripts are expressed in the Lymnaea CNS. Northern blot analysis ofLymnaea CNS poly(A⁺) RNA using a probe specifically recognizing the antisense region of the pseudo-NOS transcript identifies a prominent band of the expected size (∼2500 nt) in lane A (arrow). A less prominent transcript of ∼3200 nt is also revealed in the experiment. This suggests that there are other RNA molecules in the CNS that are antisense to the nNOS-encoding transcript. In lane B, the result of the hybridization with a probe recognizing the 3′ untranslated end of nNOS mRNA is shown. As expected, a single transcript of ∼5000 nt is revealed (arrow).

A uniquely identified neuron (the CGC) coexpresses functional NOS mRNA and pseudo-NOS RNA. InA, the results of RT-PCR experiments on RNA purified from isolated identified CGCs are illustrated. Lane 4shows that a PCR product of the expected size (598 bp) is generated by primers specific to nNOS mRNA. Similarly, in lane 2, a PCR product of the expected size (431 bp) generated from the same RNA sample by primers specific to the pseudo-NOS RNA is detected.Lanes 1 and 3 show the results of PCR experiments designed to control for possible DNA contamination of the samples analyzed in lanes 2 and 4, respectively. In these experiments, reverse transcriptase was omitted, and as a consequence, no products were generated. InB, the results from isolated B2 motoneurons are presented. Lane 4 shows a PCR product of the expected size (598 bp) generated by the same nNOS-specific primers as used inA. Note that there is no PCR product in lane 2 in which the pseudo-NOS RNA-specific primers were used.Lanes 1 and 3 represent control experiments in which reverse transcriptase was omitted. The absence of any PCR products in these lanes proves that the RNA sample used in the RT-PCR experiments was free from DNA contamination. All RT-PCR products shown have been cloned and sequenced to confirm their identity.

A schematic diagram showing the major steps of the ribonuclease protection procedure used to detect RNA–RNA duplexesin vivo. To preserve possible RNA–RNA hybrids, cytoplasmic RNA was purified from the CNS under nondenaturing conditions. To identify our hypothesized RNA–RNA duplex, the RNA has to be treated with RNase A, an enzyme that cleaves single-stranded but not double-stranded RNA molecules. After RNase A treatment, reverse transcription reactions in the presence of either P2 primer (located within the protected area) or P1 primer (located outside the protected area) are performed. After adding the P3 primer, a cDNA generated in the first reaction could be amplified using PCR and then will be revealed as a single band of the expected size by electrophoresis. In contrast, no cDNA could be produced in the second reverse transcription reaction, and subsequently, no PCR product is expected. In theleft column, the predicted results of the control experiments (no RNase A treatment) are summarized. Two RT-PCR products should be detected in the reverse transcription reaction: one generated by P2 and P3 and the other by P1 and P3.

The predicted RNA–RNA duplex exists in the CNS. The experiment was performed according to the procedure described in Figure 5. Lanes 1 and 3 show the products of RT-PCR generated by RNA treated with DNase only. Lanes 2 and 4 show the products of RT-PCR generated on RNA treated with both DNase and RNase A. In lanes 1 and2, RNA was reverse transcribed with the P2 primer located within the predicted duplex and then amplified in the presence of the P2 and P3 primers (see Figs. 3 and 5 for location of the primers). Lanes 3 and 4 show the results generated when RNA was reverse transcribed with the P1 primer located outside the predicted duplex and then amplified in the presence of P1 and P3 primers. A product of the same predicted size (139 bp) is generated by the RNA sample that was treated with DNase only and with DNase plus RNase A (lanes 1 and 2). The band shown in lane 3 is the size predicted (340 bp) of a product generated by the P1 and P3 primers. Note that there is no product in lane 4. Importantly, these results correspond precisely to those predicted in Figure 5. All of the RT-PCR products shown have been cloned and sequenced to confirm their identity.

Synthesis of nNOS protein in vitrois suppressed by the antisense pseudo-NOS RNA. Lane 1represents the result of the translation of NOS cRNA and shows the main labeled product is a protein of the expected (93 kDa) size (arrow). Lane 2 illustrates the effect of incubating the 0.2 μg of NOS cRNA with 2 μg of antisense pseudo-NOS cRNA before translation. Note strong suppression of translation of the nNOS protein. As a control for any effects on translation that are not related to the formation of a duplex, we also preincubated the 0.2 μg of NOS cRNA with a 2 μg of a sense version of the pseudo-NOS cRNA (lane 3). No inhibition of NOS protein synthesis is observed. Similar results, although weaker, have been obtained, even when the ratio between nNOS cRNA and pseudo-NOS transcripts was 2.5:1. A second major protein of ∼50 kDa can be seen in eachlane. This protein is present in the cell-free wheat germ system and performs a useful function as an internal control. Note that it is not diminished in intensity in lane 2.

Antisense RNA-mediated regulation of nNOS protein expression in vivo. A, NADPH-diaphorase staining of the buccal ganglia. A pair of symmetrical NOS-containing diaphorase-positive B2 motoneurons is indicated byarrows. B, A diagram of theLymnaea CNS showing the positions of identified neurons referred to in this investigation. Dark cell bodies of B2 motoneurons reflect the fact that the neurons are strongly and consistently NADPH-diaphorase-positive, i.e., they always contain an active nNOS protein (A, C). In contrast, <10% of all CGCs examined showed nNOS enzyme activity. To emphasize this result, the cell bodies of CGCs are shown in light color. C, A summary of our experiments on the expression of nNOS mRNA, antisense RNA, and nNOS protein in the identified neurons B2 and CGC. The asterisk indicates that nNOS protein in the CGC is not entirely absent but is detected only in ∼10% of the cells observed (n = 60).

NADPH-diaphorase staining of the cerebral ganglia.A, The majority of CGCs shows no NADPH-diaphorase activity. B, One of the very few clearly NADPH-diaphorase-positive CGCs. Cell bodies of the CGCs are indicated by arrows.