Abstract 022: A Short Open Reading Frame in the Angiotensin Type 1a Receptor 5' Leader Sequence Increases the Rate of Receptor Internalization

Background: We recently found that a seven amino acid peptide (PEP7) encoded within a short open reading frame (sORF) in exon 2 of the 5’ leader sequence (5'LS) of the angiotensin type 1a receptor (AT1aR) mRNA inhibits AT1aR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) without having any effect on the AT1aR-inositol trisphosphate protein kinase C pathway. To investigate the mechanism by which PEP7 selectively inhibits the AT1aR-Erk1/2 signaling cascade, the start codon of the sORF was mutated at adenine -108 (A-108 to T-108) to create E1,2(-108T),3-AT1aR followed by cloning the E1,2,3-AT1aR and the mutant E1,2(-108T),3-AT1aR into the pEGFP-N2 plasmid. Methods: Human embryonic kidney 293 (HEK-293) cells were transfected with E1,2,3-AT1aR (intact sORF construct) or E1,2(-108T),3-AT1aR (disrupted sORF construct) by lipofectamine. Forty eight to seventy two hours later, live cell time lapse images were collected before and after treatment with Ang II (100 nM) using a TE300 Spinning disk laser scanning confocal microscope. Image analysis was performed using Velocity software. Results: Within 5 min of Ang II stimulation, punctae formed and moved throughout the cell membrane in cells transfected with both EGFP tagged receptors; however, even though the punctae represent the localized accumulation of identical AT1aR proteins, there were distinct differences in the intensity and time course of punctae. The rate of vesicle formation after Ang II treatment was markedly decreased by disrupting the PEP7 sORF [t1/2 (s): E1,2,3-AT1aR, 203s (N=21) vs E1,2(-108T),3-AT1aR, 328s (N=14); p<0.0001]. Conclusion: The PEP7 sORF facilitates Ang II-induced AT1R internalization. These findings suggest that we have uncovered a new mechanism governing agonist-induced AT1aR cellular trafficking that could have implications not only for regulation of AT1aR signaling cascades but also for other trafficking proteins that contain an upstream sORF within their 5'LS.

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Disruption of a Short Open Reading Frame (sORF) In The mRNA 5′ Leader Sequence (5′LS) of the Type 1 Angiotensin Receptor (AT 1 R) Increases Angiotensin II (Ang II)‐ Induced AT 1 R Internalization and Signaling through the Extracellular Signal‐Regulated Kinases (ERK1/2) Pathway

The FASEB Journal ◽

10.1096/fasebj.2018.32.1_supplement.533.29 ◽

2018 ◽

Vol 32 (S1) ◽

Author(s):

Parnika Kadam ◽

Hong Ji ◽

Amrita V. Pai ◽

Robert Speth ◽

Susette Mueller ◽

...

Keyword(s):

Angiotensin Ii ◽

Leader Sequence ◽

Open Reading Frame ◽

Ang Ii ◽

Angiotensin Receptor ◽

Reading Frame ◽

Short Open Reading Frame ◽

Extracellular Signal

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A 5′-upstream short open reading frame encoded peptide regulates angiotensin type 1a receptor production and signalling via the β-arrestin pathway

The Journal of Physiology ◽

10.1113/jp270567 ◽

2015 ◽

Vol 594 (6) ◽

pp. 1601-1605 ◽

Cited By ~ 8

Author(s):

Gina L.C. Yosten ◽

Jun Liu ◽

Hong Ji ◽

Kathryn Sandberg ◽

Robert Speth ◽

...

Keyword(s):

Open Reading Frame ◽

Reading Frame ◽

Short Open Reading Frame ◽

Angiotensin Type

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An Upstream Short Open Reading Frame in the Angiotensin Type 1 Receptor Regulates the β‐Arrestin Signaling Pathway

The FASEB Journal ◽

10.1096/fasebj.2019.33.1_supplement.867.7 ◽

2019 ◽

Vol 33 (S1) ◽

Author(s):

Parnika S Kadam ◽

Hong Ji ◽

Amrita V Pai ◽

Robert Speth ◽

Susette Mueller ◽

...

Keyword(s):

Signaling Pathway ◽

Open Reading Frame ◽

Reading Frame ◽

Short Open Reading Frame ◽

Angiotensin Type

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Translation of a minigene in the 5′ leader sequence of the enterohaemorrhagic Escherichia coli LEE1 transcription unit affects expression of the neighbouring downstream gene

Biochemical Journal ◽

10.1042/bj20110912 ◽

2011 ◽

Vol 441 (1) ◽

pp. 247-253 ◽

Cited By ~ 10

Author(s):

Md. Shahidul Islam ◽

Robert K. Shaw ◽

Gad Frankel ◽

Mark J. Pallen ◽

Stephen J. W. Busby

Keyword(s):

Escherichia Coli ◽

Translation Initiation ◽

Stop Codon ◽

Leader Sequence ◽

Open Reading Frame ◽

Start Codon ◽

Target Cells ◽

Reading Frame ◽

Enterohaemorrhagic Escherichia Coli ◽

Translation Start

The 5′ end of the major RNA transcript of the LEE1 operon of enterohaemorrhagic Escherichia coli contains ~170 bases before the AUG translation start codon of the first recognized gene, ler. This unusually long leader sequence carries three potential alternative AUG start codons. Using a lac fusion expression vector, we confirmed that the ler gene AUG is functional for translation initiation, and we checked for translation initiation at the three alternative AUG codons. Whereas two of the alternative AUG codons appear incompetent for translation initiation, we detected strong initiation at the third AUG, which is followed by one AAA codon and a UAG stop codon. The location of this very short two-codon open reading frame with respect to the ler translation start appears to be critical. Hence mutations that destroy the UAG stop codon, or short deletions between the UAG stop codon and the ler translation initiation region, result in big effects on ler expression. In the context of the full-length LEE1 operon leader sequence, translation of this very short two-codon open reading frame is necessary for optimal expression of the ler gene and for the subsequent interactions of enterohaemorrhagic Escherichia coli with host target cells.

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Modulation of the rat angiotensin type 1a receptor by an upstream short open reading frame

Peptides ◽

10.1016/j.peptides.2021.170529 ◽

2021 ◽

Vol 140 ◽

pp. 170529

Author(s):

Parnika S. Kadam ◽

Susette C. Mueller ◽

Hong Ji ◽

Jun Liu ◽

Amrita V. Pai ◽

...

Keyword(s):

Open Reading Frame ◽

Reading Frame ◽

Short Open Reading Frame ◽

Angiotensin Type

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Cloning of a gar (Lepisosteus osseus) GH cDNA: trends in actinopterygian GH structure

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0160073 ◽

1996 ◽

Vol 16 (1) ◽

pp. 73-80 ◽

Cited By ~ 12

Author(s):

D A Rubin ◽

J H Youson ◽

L E Marra ◽

R M Dores

Keyword(s):

Cdna Library ◽

Untranslated Region ◽

Signal Sequence ◽

Leader Sequence ◽

Open Reading Frame ◽

Russian Sturgeon ◽

Reading Frame ◽

Terminal Codon

ABSTRACT A cDNA containing the sequence of GH was cloned and sequenced from a pituitary cDNA library for the holostean fish Lepisosteus osseus (common name: gar). The gar GH cDNA contained an open reading frame of 633 nucleotides and a 3′ untranslated region (including the terminal codon TAG) of 1058 nucleotides. The overall length of the gar GH cDNA including leader sequence, signal sequence, hormone sequence and 3′ untranslated region was 1713 nucleotides. Thus, the gar GH cDNA is the largest vertebrate GH cDNA yet cloned. A comparison of GH sequences from ancient (holostean fishes — gar and bowfin; one chondrostean fish — the Russian sturgeon) and more modern (27 species of teleosts) members of class Actinopterygii indicate that members of this class have maintained many of the invariant residues deemed necessary for GH folding motifs (intramolecular relationships) observed in mammals.

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Coding sequence and expression of the homeobox gene Hox 1.3

Development ◽

10.1242/dev.102.2.349 ◽

1988 ◽

Vol 102 (2) ◽

pp. 349-359 ◽

Cited By ~ 1

Author(s):

M. Fibi ◽

B. Zink ◽

M. Kessel ◽

A.M. Colberg-Poley ◽

S. Labeit ◽

...

Keyword(s):

Homeobox Gene ◽

Cell System ◽

T Antigen ◽

Open Reading Frame ◽

Homeodomain Protein ◽

Chromosome 11 ◽

3T3 Cells ◽

Exon 2 ◽

Reading Frame ◽

3T3 Fibroblasts

We have characterized Hox 1.3 (previously described as m2), a murine homeobox-containing gene, which is a member of the Hox 1 cluster located on chromosome 6. A cloned cDNA was isolated from an Okayama-Berg library generated from the chemically transformed cell line MB66 MCA ACL6. The protein sequence of 270 amino acids was deduced from the nucleotide sequence of an open reading frame containing the homeobox. The open reading frame is interrupted at the genomic level by a 960 bp intron and is organized in two exons. The Hox 1.3 protein was found to contain extensive sequence homology with the murine homeodomain protein Hox 2.1, which is encoded on chromosome 11. There are two homology with the regions in the first exon, i.e. a hexapeptide conserved in many homeobox-containing genes and the N-terminal domain, which was found to be homologous only to Hox 2.1. Furthermore, in exon 2 the homologies of the homeodomain regions are extended up to the carboxy terminus of Hox 1.3 and Hox 2.1. During prenatal murine development, maximal expression of Hox 1.3 is observed in 12-day embryonic tissue. The two transcripts carrying the Hox 1.3 homeobox are 1.9 kb and about 4 kb in length. An abundant Hox 1.3-specific 1.9 kb RNA is also found in F9 cells which were induced for parietal endoderm differentiation, whereas F9 teratocarcinoma stem cells do not stably express this specific RNA. Induction of the transcript occurs immediately after retinoic acid/cAMP treatment and the RNA level remains high for 5 days. Thus, the kinetics are different from the previously described homeobox transcripts Hox 1.1 and Hox 3.1. Interestingly, by analogy to the F9 cell system a negative correlation between transformation and Hox 1.3 expression is observed in 3T3 fibroblasts also. Untransformed 3T3 cells carry abundant 1.9 kb Hox 1.3 RNA, whereas the methylcholanthrene-transformed MB66 and LTK- cells or 3T3 cells transformed by the oncogenes src, fos or SV40 T antigen express only low levels.

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cis- and trans-acting suppressors of a translation initiation defect at the cyc1 locus of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/132.1.97 ◽

1992 ◽

Vol 132 (1) ◽

pp. 97-112 ◽

Cited By ~ 3

Author(s):

I Pinto ◽

J G Na ◽

F Sherman ◽

M Hampsey

Keyword(s):

Saccharomyces Cerevisiae ◽

Cytochrome C ◽

Point Mutations ◽

Start Codon ◽

Reading Frame ◽

Codon Recognition ◽

Short Open Reading Frame ◽

Initiator Codon ◽

Extragenic Suppressors ◽

Cis And Trans

Abstract The cyc1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the cyc1 transcribed leader region. We have isolated and characterized functional revertants of cyc1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF. Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from approximately 2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG. These suppressors are represented by five different genes, designated sua1-sua4 and sua6. In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting cyc1 transcription start site selection. Potential suppressor mechanisms are discussed.

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