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Survivin (BIRC5) is an anti-apoptotic protein that is important in cancer. Mechanisms responsible for controlling Survivin levels in cells include transcriptional regulation and modulation of protein stability via post-translational modifications; however to date, translational control has been poorly studied. Here, we focused particularly on the primary control elements present in the Survivin 5′ untranslated region (5′UTR). Bioinformatic analysis of ribosome occupancy on the Survivin 5′UTR revealed the presence of elongating ribosomes upstream of the canonical initiator AUG, suggesting an alternative upstream initiator AUG (uAUG) might exist. This uAUG was found out-of-frame at position −71 and appeared as a conserved element in mammals. RACE analysis revealed different transcriptional start sites for BIRC5, which indicated that translational control by this uAUG is restricted to longer 5′UTR variants. We studied the activity of the uAUG in different cell types by cloning the Survivin 5′UTR DNA sequence (wild-type and mutated variants) upstream of renilla luciferase (RLuc) into a pcDNA3 plasmid. Changes in RLuc activity were determined by luminescence assays and Western blotting. Results showed that when this uAUG was mutated to AUU or AGG in the cloned Survivin 5′UTR, RLuc activity was significantly increased. Similar results were obtained when uAUG was positioned inframe with the RLuc initiator AUG. Immunodetection of Renilla (35 kDa) by Western blotting revealed the presence of a second band (37 kDa approximately) in cells transfected with the Inframe reporter constructs, indicating that the uAUG was functional in our experimental conditions. In conclusion, our experimental data demonstrate the presence of an alternative and inhibitory initiator uAUG in the Survivin 5′ UTR. This inhibitory uAUG may help understanding how Survivin expression is downregulated under physiological or pathological conditions. • Survivin (BIRC5) is an anti-apoptotic protein that is overexpressed in most cancer types. • Primary control elements present in the Survivin 5' untranslated region are important in regulating Survivin expression. • BIRC5 is transcribed from alternative TSS to generate Survivin mRNAs with different 5'UTR sequences. • The Survivin 5'UTR contains an inhibitory out-of-frame uAUG at position −71. • These results aid in understanding how Survivin expression is controlled under physiological or pathological conditions. [ABSTRACT FROM AUTHOR]

Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear-encoded and imported into the mitochondria through post-translational or co-translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome-scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions ( UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'- UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria. [ABSTRACT FROM AUTHOR]

Background Campomelic dysplasia ( CD) is a semilethal developmental disorder caused by mutations in and around SOX9. CD is characterized by multiple skeletal malformations including bending (campomelia) of long bones. Surviving patients frequently have the acampomelic form of CD ( ACD). Methods This is a single case report on a patient with clinical and radiological features of ACD who has no mutation in the SOX9 protein-coding sequence nor a translocation with breakpoint in the SOX9 regulatory domain. We include functional studies of the novel mutant protein in vitro and in cultured cells. Results The patient was found to have a de novo heterozygous mutation c.-185G>A in the SOX9 5′ UTR. The mutation creates an upstream translation start codon, uAUG, with a much better fit of its flanking sequence to the Kozak consensus than the wild-type AUG. By in vitro transcription-translation and transient transfection into COS-7 cells, we show that the uAUG leads to translation of a short peptide from a reading frame that terminates just after the wild-type AUG start codon. This results in reduced translation of the wild-type protein, compatible with the milder phenotype of the patient. Conclusion Findings support the notion that more mildly affected, surviving CD/ ACD patients carry mutant SOX9 alleles with residual expression of SOX9 wild-type protein. Although rarely described in human genetic disease and for the first time here for CD, mutations creating upstream AUG codons may be more common than generally assumed. [ABSTRACT FROM AUTHOR]

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5'-untranslated regions (5'-UTR) due to differential splicing. The 5'-UTR variant ACD' is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication. [ABSTRACT FROM AUTHOR]

Background: Variability in protein levels is generated through intricate control of the different gene decoding phases. Presently little is known about the links between the various gene expression stages. Here we investigated the relationship between transcription and translation regulatory properties encoded in mammalian genes. Results: We found that the TATA-box, a core promoter element known to enhance transcriptional output, is associated not only with higher mRNA levels but also with positive translation regulatory features and elevated translation efficiency. Further investigation revealed general association between transcription and translation regulatory trends. Specifically, translation inhibitory features such as the presence of upstream AUG (uAUG) and increased lengths of the 5′UTR, the coding sequence and the 3′UTR, are strongly associated with lower translation as well as lower transcriptional rate. Conclusions: Our findings reveal that co-occurrence of several gene-encoded transcription and translation regulatory features with the same trend substantially contributes to the final mRNA and protein expression levels and enables their coordination. [ABSTRACT FROM AUTHOR]

Aim. The prostate tumor-inducing oncogene (PTI-1), presumably encoding a truncated form of eukaryotic translation elongation factor 1A1 (eEF1A1), was discovered as a gene overexpressed in prostate tumor samples and absent in normal tissues. The mechanism of PTI-1 oncogenicity remains obscure. Methods. Several bioinformatics methods were applied to analyze the PTI-1 mRNA structure, translation efficiency and coding potential. Results. In silico analysis of 5'UTR of its mRNA suggest that PTI-1 mRNA most probably belongs to the class of templates with low translation efficiency. Additionally, novel open reading frame (ORF) starting with alternative initiation site situated upstream of the main ORF start codon was found. Finally, the peptide that does not resemble eEF1A1 but is partially homologous to relaxin can be synthesized. Conclusions. We suggest that the alternative upstream start codon may initiate synthesis of a peptide (uPTI-1) homologous to relaxin, the hormone shown to promote the prostate cancer progression. uPTI-1 protein may interact with the respective relaxin-specific receptors, suggesting that the tumorigenic outcome of PTI-1 is possibly realized via the relaxin-dependent pathway. [ABSTRACT FROM AUTHOR]

Abstract: ADAM22 is one of three catalytically inactive ADAM family members highly expressed in the brain. Preliminary functional studies suggest possible roles in epilepsy and myelination. We report an additional eight new splice variants of human ADAM22. Analysis of the altered splicing patterns of ADAM22 mRNAs in glioma allows us to suggest alternate splicing patterns in normal brain compared to glioma may represent differential use of exon 32. We also report diversity in the 5′ leader sequences of ADAM22 mRNAs as a consequence of alternate transcriptional initiation sites. ADAM22 has an additional transcriptional initiation element producing transcripts lacking the exon 1 sequence including the signal peptide. Variable transcriptional initiation in exon 1 produces a range of ADAM22 5′ leader sequence lengths, all of which are significantly longer than those described in NCBI reference sequences. Longer 5′ leader sequences contain a second upstream AUG codon which acts to inhibit ADAM22 translation. [Copyright &y& Elsevier]

Abstract: The ADAMTS (A Disintegrin and metalloproteinase with thrombospondin-1 type repeats) family of enzymes have been implicated in turnover of extracellular matrix. We previously showed that levels of ADAMTS6 mRNA in ARPE-19 cells were markedly increased following treatment with tumour necrosis factor alpha (TNFα). This study shows that the ADAMTS6 transcript contains unusually large untranslated regions (UTRs) at both the 5′ and 3′end. The 5′UTR contains 11 AUG codons upstream of the predicted ADAMTS6 start codon and potently inhibits translation of a downstream reporter gene. However some translation can be restored by truncating the 5′UTR from the 5′end. The 5′UTR was tested for internal ribosome entry site activity using a bicistronic luciferase reporter plasmid, but none was detected. Using the 5′ and 3′UTR sequences to screen the GenBank database we identified a full length ADAMTS6 cDNA of 7262 bp. This transcript is alternatively spliced at the 3′end of the open reading frame (ORF), resulting in an extended ORF containing 3 additional tsp-1 type repeats. Quantitative RT-PCR showed that the long and short form of the ADAMTS6 ORF are co-expressed in ARPE-19 cells, but the relative levels of the two forms is modulated by TNFα. The region of the transcript encoding the catalytic domain also contains several notable differences compared to the previously published ADAMTS6 cDNA sequence, including a redefinition of the predicted active site motif. [Copyright &y& Elsevier]