A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE(2)-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-lm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.

Background:The spinocerebellar ataxia type 1 (SCA1) gene encoding ataxin-1 (ATXN1) contains an alternative open reading frame overlapping the ATXN1 coding sequence. Results: The alternative ATXN1 protein is constitutively co-expressed and interacts with ATXN1 N terminus. Alternative ATXN1 is also an RNA binding protein. Conclusion:ATXN1 is a genuine dual coding gene. Significance: Alternative ATXN1 may regulate the function of ATXN1 in physiological and pathological conditions.

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2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a flame-retardant chemical appearing at increasing concentrations and frequency in the environment and human samples. A number of health effects of exposure to BDE-47 have been observed, thyroid disruption being the most sensitive. Our objective was to examine BDE-47 interaction with thyroid receptor beta (TRβ). We used a variety of approaches, including in vitro binding assays, luciferase reporter-gene transcriptional assays, and analysis of expression of thyroid responsive genes in rat offspring exposed perinatally to BDE-47. We found that BDE-47 alone or in mixture with 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), 2,2',4,4',6-pentabromodiphenyl ether (BDE-100), and 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153) does not compete with [(125)I]T(3) for TRβ-binding even at 4000 fold higher concentrations. Also, BDE-47 does not affect thyroid responsive genes through TRβ in in vitro studies of transcription regulation. A subset of thyroid responsive genes were significantly differentially expressed in liver and frontal lobe brain samples of exposed pups, however, the action of BDE-47 was neither agonistic or antagonistic to that of thyroid hormone. We conclude that BDE-47 does not interact directly with TRβ1 nor does it influence its transcriptional activity. Developmental exposure of rats to BDE-47 leads to differential expression of thyroid responsive genes in liver and brain due to unknown mechanism.