| dc.description.abstract | Protein synthesis terminates at the first stop codon encountered by the ribosome. In stop codon readthrough, translation termination is suppressed, enabling the ribosomes to continue translation beyond the canonical stop codon upto a downstream in-frame stop codon resulting in a C-terminally extended polypeptide. Several cis- or trans-acting elements contribute to a programmed stop codon readthrough event. Evidence of stop codon readthrough and the functional significance of the readthrough isoforms have been reported for VEGFA, AGO1, AMD1, AQP4, LDH, MDH, MTCH2 and VDR. The first evidence of functionally relevant, programmed stop codon readthrough was reported in VEGFA. In the same study, genome-wide bioinformatic based analysis was done to identify other potential readthrough candidates. NNAT (encodes Neuronatin), the focus of this study, was one of the potential readthrough candidates identified.
Neuronatin (NNAT) is a small proteolipid expressed from chromosome 2 in mice and chromosome 20 in humans. The gene encoding NNAT, resides in the opposite strand within the intronic region of a larger gene, BLCAP (Bladder Cancer Associated Protein). Also known as Peg5 (Paternally Expressed Gene 5), NNAT is maternally imprinted and is thus expressed only from the paternal allele. Conservation of the proximal 3ꞌUTR of NNAT upto a second in-frame stop codon in different mammals led us to the hypothesis that NNAT mRNA undergoes stop codon readthrough. In this study, we have demonstrated readthrough in NNAT using luminescence- and fluorescence-based reporters as well as by Western blot. Analysis of previously reported ribosome profiling data coupled with the detection of endogenous NNATx (readthrough isoform) by Western blot using an antibody specific for the C-terminal extension of NNATx, confirmed stop codon readthrough in NNAT. We have also identified several cis-acting factors and an RNA-binding trans-acting factor, NONO/p54nrb, that drive stop codon readthrough in NNAT.
To understand the functional role of the readthrough isoform, NNATx, we have overexpressed the canonical (NNAT) and the readthrough (NNATx) isoforms in a mouse neuroblastoma cell line, Neuro-2a. NNAT increases cytoplasmic Ca2+ levels by inhibiting SERCA2 (Sarco/endoplasmic reticulum Ca2+-ATPase isoform 2). SERCA2, a P-type ATPase, is localized on the membrane of the endoplasmic reticulum and pumps Ca2+ from the cytoplasm to the ER. By inhibiting SERCA2, NNAT increases Ca2+ levels in the cytoplasm and promotes neuronal differentiation. Unlike NNAT, the readthrough isoform, NNATx, fails to interact with SERCA2 and does not increase cytoplasmic Ca2+ levels. As a result, NNATx does not promote neuronal differentiation.
To further understand the specific role of NNATx, we decreased the endogenous expression of NNATx using an antisense oligonucleotide (named as +43 ASO) that targets a region between 43 to 66 nucleotides in the inter stop codon region (the 3ꞌ untranslated region between the 1st and 2nd stop codons) of NNAT. With reduced expression of endogenous NNATx, +43 ASO treated cells showed increased cytoplasmic Ca2+ compared to a non-targeting oligonucleotide treated cells. Thus, by modulating stop codon readthrough of NNAT, we show that NNATx fails to promote neuronal differentiation as it fails to increase cytoplasmic Ca2+ levels.
Overall, these results demonstrate regulation of neuronal differentiation by SCR of NNAT. Importantly, this process can be exogenously regulated using a synthetic antisense oligonucleotide. | en_US |
