5?UTR sequences of the glucocorticoid receptor 1A transcript encode a peptide associated with translational regulation of the glucocorticoid receptor

Abstract

We have recently reported that glucocorticoid receptor (GR) transcript 1A, one of the five mouse GR splice variants (1A-1E), encodes membrane GR (mGR), which subsequently participates in mediating the apoptotic effects of glucocorticoids (GCs); all transcripts vary at their 5'UTR. Computer analysis of the entire1026 bp comprising the 5'UTR of transcript 1A identified five putative translation start sites at positions 85, 217, 478, 628, and 892 with the potential to encode peptides of 33, 93, 6, 18, and 41 amino acids, respectively. We then separately generated point mutations at these five upstream AUG codons of the GR 1A cDNA and performed in vitro transcription/translation experiments to investigate the regulatory effects of these sites on GR synthesis. GR translation products were immuno-captured with BUGR-2 antibody (Ab), then subjected to Western blot analysis. Mutation of the uAUG codon-2 completely inhibited GR synthesis, while mutations at the other four uAUG codons had no significant effect on the translation of transcript 1A. Antibodies (Abs) against the uORF-2 and uORF-5 protein products were used to perform Western blot analysis on cytosolic proteins from S-49 cells (which express GR transcript 1A), U937 cells transfected with GR 1A cDNA, or in vitro translation products from this cDNA. This assay identified an intense immunoreactive band of approximately 8.5 kDa recognized only with Ab to the uORF-2 peptide; this size is consistent with the computer-predicted size of the uORF-2 product, suggesting that the uORF-2 product is indeed synthesized in cells. No peptide was identified with Ab to uORF-5 peptide. Indirect fluorescent Ab staining, confocal microscopy and FACS analysis all showed that the ORF-2 peptide is localized both in the interior of the cell and at the plasma membrane. Using Ab to ORF-2 peptide for immunoadsorption we then asked whether cellular factors interact with the product of uORF-2. Immuno-captured uORF-2 peptide levels correlated with the concentrations of several salt-wash-sensitive cellular proteins, suggesting that protein-protein interactions occur between this upstream open reading frame (uORF) product and other factors. The uORF-2 product, however, does not appear to directly interact with GR, since there was no reciprocal immuno-capture between these two proteins. In summary, our results show that cells can synthesize the uORF-2 peptide, blocking of the synthesis of the uORF-2 peptide product abolishes translation of GR from the GR 1A transcript, and the peptide product of uORF-2 interacts with other cellular factors which might be involved in translation of GR.

Analysis of Small Human Proteins Reveals the Translation of Upstream Open Reading Frames of mRNAs

“…This means that there are potential short coding regions in the 5Ј-UTRs of many genes if this classical model, indeed, represents a general mechanism of translation initiation. To our knowledge, few reports have presented evidence of the translation of upstream ORFs in vivo (Diba et al 2001). Although there are also some mechanisms by which the ribosomal complex may evade the translation from the first ATG codon, such as leaky scanning (Kozak 1999) and IRES (internal ribosome entry site)-dependent translation (Meijer and Thomas 2002), we expect that the small proteins encoded by upstream ORFs in 5Ј-UTRs exist in vivo.…”

New Peptides Under the s(ORF)ace of the Genome

“…There are even examples of canonical non-coding RNAs, such as pri-miRNAs [66] and ribosomal RNAs [67][68][69], encoding biologically active peptides with well characterised functions. Similarly some uORFs have been shown to exert their regulatory function through their encoded peptides, with this regulation depending on their aa sequence [70], and being able to occur in trans [71][72][73][74].…”

Glucocorticoid receptor transcriptional isoforms and resistance in multiple myeloma cells

“…A variety of different 5 ¶ sequences may influence post-transcriptional gene processes, such as mRNA processing, export, and stability. It has also been suggested that promoter usage may regulate the differential response of GR expression to glucocorticoids (23), the expression of specific membrane or intracellular receptor isoforms (24), or even direct GR mRNA translation (25).…”