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

Abstract

Here, we show that a nitric oxide synthase (NOS) pseudogene is expressed in the CNS of the snail Lymnaea stagnalis. The pseudo-NOS transcript includes a region of significant antisense homology to a previously reported neuronal NOS (nNOS)-encoding mRNA. This suggested that the pseudo-NOS transcript acts as a natural antisense regulator of nNOS protein synthesis. In support of this, we show that both the nNOS-encoding and the pseudo-NOS transcripts are coexpressed in giant identified neurons (the cerebral giant cells) in the cerebral ganglion. Moreover, reverse transcription-PCR experiments on RNA isolated from the CNS establish that stable RNA-RNA duplex molecules do form between the two transcripts in vivo. Using an in vitro translation assay, we further demonstrate that the antisense region of the pseudogene transcript prevents the translation of nNOS protein from the nNOS-encoding mRNA. By analyzing NOS RNA and nNOS protein expression in two different identified neurons, we find that when both the nNOS-encoding and the pseudo-NOS transcripts are present in the same neuron, nNOS enzyme activity is substantially suppressed. Importantly, these results show that a natural antisense mechanism can mediate the translational control of nNOS expression in the Lymnaea CNS. Our findings also suggest that transcribed pseudogenes are not entirely without purpose and are a potential source of a new class of regulatory gene in the nervous system.

Eukaryotic regulatory RNAs: an answer to the ‘genome complexity’ conundrum

“…Transcription of a pseudogene that is homologous to the neuronal nitric oxide synthase (nNOS) gene in a population of neurons decreases the expression levels for the nNOS gene. Interestingly, RNA isolated from these neuronal cells confirmed the presence of an in vivo stable RNA-RNA duplex between these two transcripts suggested to arise via a reversecomplement sequence found at the 5Ј end of the pseudo-NOS transcript (Korneev et al 1999). This study implicated the pseudo-NOS transcript as a natural antisense regulator of nNOS protein synthesis (Korneev et al 1999).…”

“…A role of pseudogene transcripts in gene regulation has also been reported in the snail Lymnea stagnalis (Korneev et al 1999(Korneev et al , 2005. Transcription of a pseudogene that is homologous to the neuronal nitric oxide synthase (nNOS) gene in a population of neurons decreases the expression levels for the nNOS gene.…”

A Trim5-cyclophilin A fusion protein found in owl monkey kidney cells can restrict HIV-1

“…Although the process of retrotransposition may have been necessary for genome evolution (36), the vast majority of existing pseudogenes are considered as ''junk'' DNA. Although some are transcribed, many have accumulated mutations that would preclude function, and only a very small number may play physiological roles (37)(38)(39). Indeed, even though exon shuffling may have been of great importance in the evolutionary past (40,41), the Trim5-CypA protein appears to belong to a tiny handful of functional proteins unambiguously created in this manner.…”

ROSINA (RSI) is part of a CACTA transposable element, TamRSI, and links flower development to transposon activity

“…PCR ampliWcation of products longer than 4.5 kb were ampliWed with the Expand High Fidelity system (ROCHE), using ampliWcation programs as indicated in the manufacturer's instructions. Doublestranded RNA was detected according to the protocol described in Korneev (1999). The list of the primers used in this study is found in supplementary Materials and methods, Table 1.…”

“…The smear at the bottom of the Southern blot is likely due to breakdown of the radioactively labelled Wrst strand during PCR and/or linear ampliWcation of breakdown products which have lost one primer-annealing site, but which still can hybridise to the radioactive probe Parts of RSI RNA and the TNP RNA form double-stranded RNA The detection of the RSI-ATs suggested that doublestranded RNAs (dsRNA) might form in cells where both RSI and RSI-ATs transcripts are expressed. To detect dsRNA we took advantage of the methods described in Korneev et al (1999), which exploited the feature of the RNase-ONE enzyme to degrade single-stranded RNA leaving dsRNA intact. Total RNA was extracted under non-denaturing conditions, treated with an excess of RNase-ONE and reverse-transcribed with primers located outside and inside the hypothetical dsRNA molecules (Fig.…”