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Gene Expression Regulation by Upstream Open Reading Frames In Silva J, Fernandes R, Romão L. J Rare Dis Res Treat. (2017) 2(4): 33-38 Journal of www.rarediseasesjournal.com Rare Diseases Research & Treatment Mini review Open Access Gene expression regulation by upstream open reading frames in rare diseases Joana Silva1,2, Rafael Fernandes1,2, and Luísa Romão1,2* 1Department of Human Genetics, Instituto Nacional de Saúde Doutor Ricardo Jorge, Lisboa, Portugal 2Gene Expression and Regulation Group, Biosystems & Integrative Sciences Institute (BioISI), Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal Article Info ABSTRACT Article Notes Upstream open reading frames (uORFs) constitute a class of cis-acting Received: June 19, 2017 elements that regulate translation initiation. Mutations or polymorphisms that Accepted: July 21, 2017 alter, create or disrupt a uORF have been widely associated with several human *Correspondence: disorders, including rare diseases. In this mini-review, we intend to highlight the Dr. Luísa Romão, Department of Human Genetics, Instituto mechanisms associated with the uORF-mediated translational regulation and Nacional de Saúde Doutor Ricardo Jorge, Av. Padre Cruz, describe recent examples of their deregulation in the etiology of human rare 1649-016, Lisboa, Portugal; Tel: (+351) 21 750 8155; Fax: diseases. Additionally, we discuss new insights arising from ribosome profiling (+351) 21 752 6410; studies and reporter assays regarding uORF features and their intrinsic role E-mail: [email protected]. in translational regulation. This type of knowledge is of most importance to © 2017 Romão L. This article is distributed under the terms of design and implement new or improved diagnostic and/or treatment strategies the Creative Commons Attribution 4.0 International License. for uORF-related human disorders. Keywords Upstream open reading frame (uORF) Non-AUG initiation codon Translation initiation Introduction Translational regulation Over the past few years, many genome-wide studies [RNA deep Human rare disease Stress sequencing, ribosome profiling (RiboSeq), mass spectrometry- based methodologies] pointed out translation as a major regulator of gene expression, being recognized as a 1–9key post-transcriptional mechanism by which cells rapidly change their expression patterns in response to a wide variety of stimuli . RiboSeq is the most promising genome-wide5,10 approach. to monitor in vivo translation, providing also new information about mechanisms of protein synthesis and its control Translation is a tightly controlled process that comprises four different steps:11 initiation, elongation, termination and ribosome recycling. Translation initiation is the most regulated step of translation . In eukaryotes, translation initiation starts with the eIF4G,recruitment to the of mRNA the cap-binding 5’ end. The protein unwinding complex, of namelythe 5’UTR eukaryotic by the initiation factor 4F (eIF4F), which comprises eIF4E, eIF4A and helicase eIF4A, enables binding of the 40S ribosomal subunit. The association of eIF1, eIF1A and eIF3 to the 40S subunit facilitates the binding of the ternary complex eIF2-GTP-Met-tRNAi. The resulting 43S preinitiation complex can land next to the cap and scans in a 5’ to 3’direction until it recognizes an initiation codon base-pairing with methionine initiator-tRNA (Met-tRNAi). Upon recognition of the start codon, eIF5 stimulates GTP hydrolysis, resulting in the release of eIF2-GDP and probably of other 40S-bound11 initiation factors. eIF5B catalyzes the joining of 60S subunit to form an 80S ribosome, and elongation can start (reviewed in ). Page 33 of 38 Silva J, Fernandes R, Romão L. J Rare Dis Res Treat. (2017) 2(4): 33-38 Journal of Rare Diseases Research & Treatment ORF cannot occur1,30 There are several cis-acting elements involved in the In these circumstances, translation reinitiation at the main regulation of translation1,11 initiation, for instance, internal . An efficient translation repression ribosome entry sites (IRESs) and upstream open reading mediated by translatable uORF(s) is positively correlated frames (uORFs) . IRESs are highly structured RNA with: (i) a strong uAUG context, (ii) a large distance from sequences that allow the recruitment of the 40S ribosomal the 5’ cap to the uAUG, (iii) a great number of uORFs, (iv) mechanismsubunit directly to the initiation codon or to its vicinity, thea long uORF-encoded uORF, and (v) peptide a short can distance exert an23,31–33between inhibitory the uORFeffect apromoting sequence 12,13beginningtranslation at initiation an initiation via codon,a cap-independent within the 5’ and the main coding sequence (CDS) . Additionally, . On the other hand, a uORF is defined as a termination codon positioned upstream or downstream ribosomesin translation in athrough sequence-dependent a potential interaction manner or/and with the in untranslated region (5’UTR) of a transcript, in frame 1,14,15with. translational machinery by stalling the translating (overlapped uORF) of the main ORF initiation codon an indirect34–36 way through interactions with other small uORFs, the most abundant and the best understood class of molecules . Moreover, . the uORF-encoded peptides can small ORFs (sORFs), are sequences that encode for peptides have additional biological37 functions in the cell, working as up to 10016,17 amino acids, and play different biological. These roles - trans-regulatory factors cis 11 in the cell . uORFs are typically described18–22 as repressors The cell microenvironment influences the recognition of translation initiation at the main ORF of the AUG initiation codon by the 43S PIC . During regulatory elements are prevalent genome-wide being estimated that approximately half of the human transcripts stress conditions, such as hypoxia, endoplasmic reticulum contain at least one uORF, and many of them are conserved kinases(ER) stress or nutrient depletion, eIF2α subunit is phosphorylated38–40 at serine 51 (eIF2α-P) by specific among species, suggesting18,23–27 an evolutionary selection of functional uORFs . Genes that need a highly controlled . This phosphorylation prevents eIF2 recycling translational regulation, such as oncogenes and genes by the guanine nucleotide exchange factor eIF2B, thus impairing the formation11,31,41 of the ternary complex and uORFsinvolved in. Thus,cell growth, it is easy differentiation, to understand developmentthat mutations and or response to stress reducing the global rate of translation as part of the cell stress 1,14,23response, are the typical classes of genes harboring . However, facing this global translational repression a group of transcripts escape polymorphisms that disrupt, create1,14 or modify uORFs can potentially be associated with the development of several and increase their translational rates via uORF-mediated mechanisms, specifically3,32,38–41 the ones that are involved in disorders, including rare diseases . Additionally, uORFs. For cell stress-response . In the context of high levels and IRESs in the same transcript can cooperate to regulate12 protein synthesis, although with an antagonist effect codonof eIF2α-P,31 uORFs are usually bypassed by the scanning ribosome that will then access the main AUG initiation example, translation of fibroblast. growth factor 9 (FGF9) is repressed by a uORF in physiological28 conditions, and . This phenomenon is called leaky scanning and induced by a IRES in hypoxia is responsible for allowing expression of, GADD34for instance, and reporter assays, here, we review the mechanisms about proteins involved in the ER stressCHOP response, like the growth By including the new contributions from RiboSeq analyses arrest and DNA damage-inducible protein ( ). Theand the C/EBP homologous protein ( ), which are3,20,21 encoded uORF-mediated translational regulation, and show how potentiatedby mRNAs with by a twoweak and uAUG one sequence uORFs, respectively context . uORFstheir deregulation and translational can cause human regulation rare disorders. leaky scanning process in these two transcripts3,20,21,32,42 is mainly its initiation codon needs to be recognized. This process For a uORF to function as a translational regulator In other cases, translation GCN4reinitiation at the main ORF occurs after translationATF4 of at least one uORF. The therequires scanning the recruitment of the 5’UTR of the and 43S the pre-initiation recognition complex of the yeast general control protein ( ) and the activating (PIC) to the mRNA 5’-cap that, as mentioned above, allows transcription factor 4 ( ), with four and two uORFs in in11 their 5’UTRs, respectively, are good examples. In ofamino transcripts acid upstream AUG (uAUG) codon to start translation (reviewed encoding stress related-proteins 22,43whoseGCN4 expression ). When the ribosome reaches the uORF stop codon, it depends on translation reinitiation can either: (i) dissociate and be recycled, which induces thestarvation ribosome conditions, to bypass the the first other uORF uORFs, of granting is efficiently the time translational repression of the downstream ORF(s), or (ii) translated, but low levels of available ternary complex force codonthe 40S subunit does not dissociate from the mRNA and 43. In the case of nonsense-mediatedis able1,14,22,25,29 to reinitiate translationmRNA decay, at ifa thedownstream uORF stop initiation codon is ATF4to acquire a newly formed ternary complex and reinitiate recognized as .a Translationpremature of a uORF may also trigger1. translation at the main initiation codon , only the first uORF is translated in stress
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