Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans

Calvo, Sarah E.; Pagliarini, David J.; Mootha, Vamsi K.

doi:10.1073/pnas.0810916106

Cited by 798 publications

(966 citation statements)

References 49 publications

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“…These percentages are similar to those reported in previous studies [16,24]. Next, we divided the transcripts of each dataset into four subgroups for comparisons: (1) transcripts without uAUGs (G 0 ); (2) transcripts with only alternative uORF(s) (G a ); (3) transcripts with only strict uORF(s) (G s ; their uAUGs are designated as "SuAUGs"); (4) transcripts with only overlapping uORF(s) (G v ; their uAUGs are designated as "VuAUGs").…”

Section: Resultssupporting

confidence: 93%

Exploring the selective constraint on the sizes of insertions and deletions in 5' untranslated regions in mammals

2011

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BackgroundSmall insertions and deletions ("indels" with size ≦ 100 bp) whose lengths are not multiples of three (non-3n) are strongly constrained and depleted in protein-coding sequences. Such a constraint has never been reported in noncoding genomic regions. In 5'untranslated regions (5'UTRs) in mammalian genomes, upstream start codons (uAUGs) and upstream open reading frames (uORFs) can regulate protein translation. The presence of non-3n indels in uORFs can potentially disrupt the functions of these regulatory elements. We thus hypothesize that natural selection disfavors non-3n indels in 5'UTRs when these regulatory elements are present.ResultsWe design the Indel Selection Index to measure the selective constraint on non-3n indels in 5'UTRs. The index controls for the genomic compositions of the analyzed 5'UTRs and measures the probability of non-3n indel depletion downstream of uAUGs. By comparing the experimentally supported transcripts of human-mouse orthologous genes, we demonstrate that non-3n indels downstream of two types of uAUGs (alternative translation initiation sites and the uAUGs of coding sequence-overlapping uORFs) are underrepresented. The results hold well regardless of differences in alignment tool, gene structures between human and mouse, or the criteria in selecting alternatively spliced isoforms used for the analysis.ConclusionsTo our knowledge, this is the first study to demonstrate selective constraints on non-3n indels in 5'UTRs. Such constraints may be associated with the regulatory functions of uAUGs/uORFs in translational regulation or the generation of protein isoforms. Our study thus brings a new perspective to the evolution of 5'UTRs in mammals.

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Section: Resultssupporting

confidence: 93%

Exploring the selective constraint on the sizes of insertions and deletions in 5' untranslated regions in mammals

2011

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“…After removing 2 out of 3 uAUG codons, of which one was predicted to create an overlapping open reading frame that could potentially interfere with translation from the canonical PTCH1b ORF and thus reduce the PTC1-L levels, luciferase activity was restored to the level observed with the 188nt-long PTCH1b 5'UTR. 54 The introduced mutations did not affect the levels of Fluc mRNA, indicating that the observed difference in luciferase activity must be ascribed to post-transcriptional control.…”

Section: Discussionmentioning

confidence: 95%

Regulation of humanPTCH1bexpression by different 5' untranslated regioncis-regulatory elements

et al. 2015

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Inga (2015) Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements, RNA Biology, 12:3, 290-304, DOI: 10.1080/15476286.2015 PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG) n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

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“…Still, the production from these transcripts of low levels of oncoproteins that might be enough for cellular transformation can not be completely ruled out, as some non-sense transcripts are known to evade the NMD pathway, 20 and transcripts containing upstream ORFs can be translated at low levels. 21 Also, the possibility for oncogenic growth triggered by fusion transcripts between the viral vector and non-coding cellular genomic sequence exists. Many microRNAs, long non-coding RNAs and other non-coding RNAs with regulatory functions are encoded in introns and intergenic genomic regions.…”

Section: Lentiviral Vector Transcriptional Read-through In Keratinocytesmentioning

confidence: 99%

Risk assessment in skin gene therapy: viral–cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors

et al. 2011

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Cutaneous gene therapy can be envisioned through the use of keratinocyte stem cell clones in which retroviral genotoxic risks can be pre-assessed. While transactivation of cellular genes by the retroviral long terminal repeat enhancer has been proven in experimental and clinical settings, the formation of chimeric viral-cellular transcripts originated by the inefficient termination (read-through) of retroviral transcripts remains to be studied in depth. We now demonstrate the widespread presence of viral-cellular fusion transcripts derived from integrated proviruses in keratinocytes transduced with self-inactivating (SIN) retroviral vectors. We have detected high molecular weight RNAs in northern blot analysis of retroviral vector expression in individual cell clones. Characterization of some of these transcripts revealed that they originate from genes located at the proviral integration sites. One class of transcripts corresponds to fusions of the viral vectors with intronic sequences, terminating at cryptic polyadenylation sites located in introns. A second class comprises fusion transcripts with coding sequences of genes at the integration sites. These are generated through splicing from a cryptic, not previously described donor site in the lentiviral vectors to exons of cellular genes, and have the potential to encode unintended open reading frames, although they are downregulated by cellular mechanisms. Our data contribute to a better understanding of the impact of SIN lentiviral vector integration on cellular gene transcription, and will be helpful in improving the design of this type of vectors.

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Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans

Cited by 798 publications

References 49 publications

Exploring the selective constraint on the sizes of insertions and deletions in 5' untranslated regions in mammals

Exploring the selective constraint on the sizes of insertions and deletions in 5' untranslated regions in mammals

Regulation of humanPTCH1bexpression by different 5' untranslated regioncis-regulatory elements

Risk assessment in skin gene therapy: viral–cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors

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