An RNA stem-loop functions in conjunction with an upstream open reading frame to direct preferential translation in the integrated stress response

Abstract

In response to environmental stresses, cells invoke translational control to conserve resources and rapidly reprogram gene expression for optimal adaptation. A central mechanism for translational control involves phosphorylation of the α subunit of eIF2 (p-eIF2α), which reduces delivery of initiator tRNA to ribosomes. Because p-eIF2α is invoked by multiple protein kinases, each responding to distinct stresses, this pathway is named the integrated stress response (ISR). While p-eIF2α lowers bulk translation initiation, many stress-related mRNAs are preferentially translated. The process by which ribosomes delineate gene transcripts for preferential translation is known to involve upstream open reading frames (uORFs) embedded in the targeted mRNAs. In this study, we used polysome analyses and reporter assays to address the mechanisms directing preferential translation of human IBTKα in the ISR. The IBTKα mRNA encodes four uORFs, with only 5′-proximal uORF1 and uORF2 being translated. Of importance, the 5′-leader of IBTKα mRNA also contains a phylogenetically conserved stem-loop of moderate stability that is situated 11 nucleotides downstream of uORF2. The uORF2 is well translated and functions in combination with the stem-loop to effectively lower translation reinitiation at the IBTKα coding sequence. Upon stress-induced p-eIF2α, the uORF2/stem loop element can be bypassed to enhance IBTKα translation by a mechanism that may involve the modestly translated uORF1. Our study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as the "bar code" by which scanning ribosomes can delineate which mRNAs are preferentially translated in the ISR. In response to environmental stresses, cells invoke translational control to conserve resources and rapidly reprogram gene expression for optimal adaptation. A central mechanism for translational control involves phosphorylation of the α subunit of eIF2 (p-eIF2α), which reduces delivery of initiator tRNA to ribosomes. Because p-eIF2α is invoked by multiple protein kinases, each responding to distinct stresses, this pathway is named the integrated stress response (ISR). While p-eIF2α lowers bulk translation initiation, many stress-related mRNAs are preferentially translated. The process by which ribosomes delineate gene transcripts for preferential translation is known to involve upstream open reading frames (uORFs) embedded in the targeted mRNAs. In this study, we used polysome analyses and reporter assays to address the mechanisms directing preferential translation of human IBTKα in the ISR. The IBTKα mRNA encodes four uORFs, with only 5′-proximal uORF1 and uORF2 being translated. Of importance, the 5′-leader of IBTKα mRNA also contains a phylogenetically conserved stem-loop of moderate stability that is situated 11 nucleotides downstream of uORF2. The uORF2 is well translated and functions in combination with the stem-loop to effectively lower translation reinitiation at the IBTKα coding sequence. Upon stress-induced p-eIF2α, the uORF2/stem loop element can be bypassed to enhance IBTKα translation by a mechanism that may involve the modestly translated uORF1. Our study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as the "bar code" by which scanning ribosomes can delineate which mRNAs are preferentially translated in the ISR. Translational control is a rapid and efficient process by which cells reprogram gene expression. This control process involves modulation of translation factors and regulatory elements embedded in gene transcripts, which together determine translational efficiencies genome-wide. An important mechanism in translational control features phosphorylation of the α subunit of eukaryotic initiation factor 2 (p-eIF2α). Environmental stresses induce p-eIF2α, which lowers coupling of eIF2 with GTP that is required for delivery of initiator Met-tRNAiMet to ribosomes for recognition of start codons (1Wek R.C. Role of eIF2alpha kinases in translational control and adaptation to cellular stress.Cold Spring Harb. Perspect. Biol. 2018; 10a032870Crossref PubMed Scopus (234) Google Scholar). The ensuing reduction in bulk translation initiation conserves energy and nutrients and helps cells facilitate reprogramming toward adaptive gene expression. Because multiple protein kinases direct p-eIF2α and translational control in response to diverse stresses, this signaling pathway is referred to as the integrated stress response (ISR) (2Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. et al.An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2464) Google Scholar). While the ISR lowers global translation initiation, the translation efficiency of many mRNAs is not changed or is enhanced in response to p-eIF2α. In this way, there is a gradient of translation efficiencies controlled by p-eIF2α, ranging from repressed, resistant, or preferential (1Wek R.C. Role of eIF2alpha kinases in translational control and adaptation to cellular stress.Cold Spring Harb. Perspect. Biol. 2018; 10a032870Crossref PubMed Scopus (234) Google Scholar, 3Baird T.D. Palam L.R. Fusakio M.E. Willy J.A. Davis C.M. McClintick J.N. et al.Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha.Mol. Biol. Cell. 2014; 25: 1686-1697Crossref PubMed Scopus (83) Google Scholar, 4Starck S.R. Tsai J.C. Chen K. Shodiya M. Wang L. Yahiro K. et al.Translation from the 5' untranslated region shapes the integrated stress response.Science. 2016; 351: aad3867Crossref PubMed Scopus (230) Google Scholar, 5Andreev D.E. O'Connor P.B. Fahey C. Kenny E.M. Terenin I.M. Dmitriev S.E. et al.Translation of 5' leaders is pervasive in genes resistant to eIF2 repression.Elife. 2015; 4e03971Crossref PubMed Scopus (223) Google Scholar). A central gene transcript that is preferentially translated in response to p-eIF2α is ATF4 (CREB2), encoding a transcriptional activator of the ISR (1Wek R.C. Role of eIF2alpha kinases in translational control and adaptation to cellular stress.Cold Spring Harb. Perspect. Biol. 2018; 10a032870Crossref PubMed Scopus (234) Google Scholar, 2Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. et al.An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2464) Google Scholar, 6Vattem K.M. Wek R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.Proc. Natl. Acad. PubMed Scopus Google Scholar). of ATF4 mRNA translation in response to p-eIF2α by a mechanism that features upstream open reading frames (uORFs) the ATF4 coding K.M. Wek R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.Proc. Natl. Acad. PubMed Scopus Google Scholar, H.P. Novoa I. Zhang Y. Zeng H. Wek M. et translation initiation stress-induced gene expression in mammalian Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The 5′-proximal uORF1 is a element that ribosomes to be the ATF4 mRNA and scanning for reinitiation at a downstream coding K.M. Wek R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.Proc. Natl. Acad. PubMed Scopus Google Scholar, Wek R.C. open reading frames translation in the integrated stress Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). In the of there is of p-eIF2α and scanning ribosomes to rapidly at the coding uORF2. The uORF2 is a element that is situated with the ATF4 and reduces ATF4 of p-eIF2α reduces eIF2 and a of the scanning ribosomes to the uORF2 and translation at the ATF4 K.M. Wek R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.Proc. Natl. Acad. PubMed Scopus Google Scholar, Wek R.C. open reading frames translation in the integrated stress Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). of ATF4 protein of ISR genes in amino acid and with of factors as also known as cellular and of p-eIF2α (2Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. et al.An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2464) Google Scholar, H.P. Novoa I. Zhang Y. Zeng H. Wek M. et translation initiation stress-induced gene expression in mammalian Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, I. Zeng H. H.P. of the protein response by of Biol. PubMed Scopus Google Scholar, and protein a signaling protein and Biol. PubMed Scopus Google Scholar, Y. of a regulatory loop that protein translation during Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, et transcriptional protein to Biol. PubMed Scopus Google Scholar, Wek Lu H.P. et factor is to the eukaryotic initiation factor 2 stress Biol. PubMed Scopus Google Scholar). with is to preferential translation during p-eIF2α by a mechanism involving scanning a that downstream translation Wek R.C. open reading frames translation in the integrated stress Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, Willy J.A. C. Wek R.C. reinitiation translation and regulates the integrated stress Biol. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). In each of the preferential translation uORFs serve as a critical "bar code" scanning ribosomes delineate which mRNAs are translated during the ISR T.D. Wek R.C. initiation factor 2 phosphorylation and translational control in Full Text Full Text PDF PubMed Scopus Google Scholar). The of the uORFs, coding efficiency of translation initiation, and and in the 5′-leader of the are as critical for ribosomes to at downstream coding Wek R.C. open reading frames translation in the integrated stress Biol. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, upstream open reading that control PubMed Scopus Google Scholar, Translational control by of eukaryotic 2016; PubMed Scopus Google Scholar). there that can to the translation efficiencies in the that in the 5′-leader of mRNAs can scanning and start during stresses Y. et mRNA translation during integrated stress Cell. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). RNA structures can and scanning to ISR translation are Translational control by of eukaryotic 2016; PubMed Scopus Google Scholar, M. and mechanisms of of translation by secondary in Biol. PubMed Scopus Google Scholar). In this study, we preferential translation of the ISR gene encoding a protein that may targeted protein L. C. et of and PubMed Scopus Google Scholar, C. et regulates the to Biol. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. et of of in and the protein Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). we that IBTKα is preferentially translated in response to p-eIF2α during stress T.D. Palam L.R. Fusakio M.E. Willy J.A. Davis C.M. McClintick J.N. et al.Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha.Mol. Biol. Cell. 2014; 25: 1686-1697Crossref PubMed Scopus (83) Google Scholar). The 5′-leader of mammalian IBTKα mRNA features four uORFs, and is that or of may serve as elements in translation T.D. Palam L.R. Fusakio M.E. Willy J.A. Davis C.M. McClintick J.N. et al.Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha.Mol. Biol. Cell. 2014; 25: 1686-1697Crossref PubMed Scopus (83) Google Scholar). we that a critical of preferential translation of IBTKα involves of which downstream reinitiation at the IBTKα a phylogenetically conserved RNA secondary that is situated downstream of the uORF2. that RNA secondary structures can in conjunction with uORFs to serve as critical elements of the by which scanning ribosomes delineate preferential translation of gene transcripts in the ISR. IBTKα in a for gene transcripts that are preferentially translated in response to stress T.D. Palam L.R. Fusakio M.E. Willy J.A. Davis C.M. McClintick J.N. et al.Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha.Mol. Biol. Cell. 2014; 25: 1686-1697Crossref PubMed Scopus (83) Google Scholar). A of the 5′-leader of the IBTKα that there are four upstream uORFs in human and with the 5′-proximal uORF1 and uORF2 conserved of IBTKα gene transcripts that conserved the is RNA secondary stem-loop of that is situated 11 nucleotides downstream of the uORF2 A and address the mechanisms human IBTKα expression during ISR and the of the stem-loop and the uORFs in the translational control of human we the in the IBTKα gene in human cells The and cells with the stress or and of IBTKα protein by Upon cells enhanced of IBTKα protein that by of the ISR and the cells of IBTKα protein of with that IBTKα gene is by ATF4 in the ISR T.D. Palam L.R. Fusakio M.E. Willy J.A. Davis C.M. McClintick J.N. et al.Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha.Mol. Biol. Cell. 2014; 25: 1686-1697Crossref PubMed Scopus (83) Google we of IBTKα mRNA as by in the and cells stress that expression of IBTKα is by stress and of the to of IBTKα protein expression of the stress regulatory elements are of human IBTKα of of IBTKα uORFs and RNA mammalian the uORFs to and the IBTKα for The is downstream of uORF2. The and of conserved in the IBTKα with a of The and the energy of a RNA secondary The RNA PubMed Scopus Google Scholar). cells and the IBTKα with or for and the of and by of IBTKα protein are to a IBTKα used as a in the and IBTKα cells with or for and of IBTKα mRNA by and are as a that are to cells with with to and the IBTKα cells with or for and by polysome the of the cells are with and The polysome to of of cells for or is and and RNA from the polysome of and and the of IBTKα transcript from of and cells by as in the and the of ATF4 and transcripts also from the The the of and ATF4 mRNAs in each and toward polysome during stress The IBTKα mRNA toward and in response to with a in with stress for ATF4 and transcripts toward and with stress coding human upstream open reading translational control of IBTKα polysome the and cells with or and and to gradient to well translated from and ribosomes. stress and p-eIF2α translation initiation as by a reduction in of human IBTKα mRNA in the gradient from the cells a toward during of preferential translation during bulk translation This toward in response to stress to ATF4 and mRNA and known to be to translational and transcriptional during the ISR (1Wek R.C. Role of eIF2alpha kinases in translational control and adaptation to cellular stress.Cold Spring Harb. Perspect. Biol. 2018; 10a032870Crossref PubMed Scopus (234) Google Scholar). is that there a of IBTKα mRNA toward the in response to there that not for ATF4 and transcripts toward with In cells IBTKα there of the IBTKα transcript with of with only a toward the polysome in response to ATF4 mRNA from IBTKα cells a toward in response to stress the that IBTKα is to preferential translation in the and the embedded in the 5′-leader of IBTKα mRNA a that can be at in during delineate the regulatory elements directing the preferential translation of we a translational reporter that a the IBTKα and the This reporter cells in combination with a encoding to in in this Upon the with in the reporter mRNA as by is that the by stress is a and the the bulk translation that p-eIF2α during the reduction in for a of the in the of in response to the of a of C. Zhang K. et of a with and PubMed Scopus Google expression in response to the of this and the ISR in translation that the reporter can features of preferential translation during the ISR. are four uORFs in the human IBTKα and we to determine each is translated by direct to the The uORF2 is with translation of uORF1 and translation of uORFs and are with the that uORF1 and uORF2 are conserved mammalian IBTKα the of each in the reporter the 5′-leader the of each in IBTKα translation the initiation to of uORF1 to to induce in response to stress of uORF2 to expression in the of stress that uORF1 can be a element in IBTKα translational uORF2 is a that there of uORF1 and uORF2 in the reporter that of the uORF2 is in translational control This also that the of uORF1 is in to uORF2. of and or in combination not of in response to stress as to and are with the translation for uORFs and and that elements are not to IBTKα translational the of the 5′-proximal uORF1 can translation of the uORF2. The uORF2 to the in the or of uORF1. of which of uORF1 and is that mRNA for also during the stress that in transcript a to this that of the the 5′-proximal uORF1 is a element is that translation of the element can in translation of the a element that is to reinitiation of translation at the downstream IBTKα Our of of the embedded in the 5′-leader of IBTKα that a in the translational control this in the reporter by targeted of the element and of in the of stress of a stem-loop of stability to of a at this is central for the IBTKα translation control the by is a of the four uORFs in the only the in a in expression to a to An reporter that the four uORFs in combination with a of the to in to the reporter only the uORFs to that the by is not of scanning and IBTKα the is from to the uORF2. of uORF2 to of expression in the of stress of the to in that only modestly with the of uORF2 and the that the elements in combination to the downstream IBTKα translation address the of the to we the 5′-leader of the reporter that the nucleotides downstream of the uORF2 the the and uORF2 to of during the as to the of to during of nucleotides the and uORF2 not enhance that for the that the of the to the uORF2 is critical for functions in IBTKα translational control in that the to the functions of the we that the to the of uORF2 to translation reinitiation at the IBTKα this reporter we the from the reporter and expression in the of stress This the that the functions to reinitiation translation of uORF2. we that the in the uORF2 situated in the 5′-leader of is the for downstream translation reinitiation at the downstream Willy J.A. C. Wek R.C. reinitiation translation and regulates the integrated stress Biol. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). In that study, we that the of translation of the uORF2 the of ribosomes to be the mRNA and scanning downstream for translation of the Willy J.A. C. Wek R.C. reinitiation translation and regulates the integrated stress Biol. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). Of importance, the element is and can be uORFs to this Willy J.A. C. Wek R.C. reinitiation translation and regulates the integrated stress Biol. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). this we the the uORF2 in the of the and that this with translation reinitiation of the uORF2 in this reporter for this uORF2 the of initiation to of of stress The reporter for the to the of the uORF2 to reinitiation of translation a reporter only the uORF2 and upstream of the of the uORF2 or the to of to the and to with the of the the uORF2 in the reporter of the only of expression of the initiation of the uORF2 the to that of the or the important for uORF2 to downstream reinitiation of we is to the of to translation reinitiation at this the uORF1 in which is a coding that is well translated and for efficient reinitiation of translation at downstream coding K.M. Wek R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.Proc. Natl. Acad. PubMed Scopus Google Scholar, H.P. Novoa I. Zhang Y. Zeng H. Wek M. et translation initiation stress-induced gene expression in mammalian Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In this reporter we the uORF2 by of the initiation from to and the for a of reinitiation of the the reporter a and of the uORF1 of the enhanced that the by is not downstream of uORF1 for the elements to translation that the is and can lower translation initiation downstream in with A critical element of the of mRNAs is uORFs that serve to direct the translation efficiencies in response to p-eIF2α. In this study, we to the preferential translation of human IBTKα mRNA during four uORFs, uORF2 to be well translated and serve as element that translation reinitiation at the downstream IBTKα in the uORF2 encodes only a and by not to be efficient of uORF2 functions in conjunction with a stem-loop that is 11 nucleotides downstream and together elements are critical for downstream the uORF2 and the are well conserved the that in IBTKα translational The IBTKα is and can be downstream of the uORF1 in the ATF4 mRNA to from that for efficient translation reinitiation to that downstream that the translational control can uORFs and RNA secondary which can in combination to translation efficiencies during the ISR. with IBTKα uORF1 is modestly and is to to that translation of uORF1 initiation at the uORF2 may be a to the of uORF2 that is to in response to embedded in of mRNAs can efficiencies of translation initiation Translational control by of eukaryotic 2016; PubMed Scopus Google Scholar). While stem-loop structures can scanning of RNA structures of as that for IBTKα not to with the initiation process M. and mechanisms of of translation by secondary in Biol. PubMed Scopus Google Scholar). are RNA stem-loop structures downstream of the start can the scanning to enhanced for ribosomes to initiation codons in upstream open reading that control PubMed Scopus Google Scholar, Translational control by of eukaryotic 2016; PubMed Scopus Google Scholar, J.A. C.M. Wang C. et conserved uORFs mRNA translation to start Natl. Acad. Scopus Google Scholar, M. secondary recognition of initiator codons by eukaryotic Natl. Acad. PubMed Scopus Google Scholar). ribosomes can with scanning which in of the and translation at start In the of the in IBTKα the of the uORF2 start is optimal and the reporter of the is well which the that the to enhance scanning recognition and translation of the uORF2. RNA structures are also for to facilitate translation initiation of the of mRNAs The mechanism of eukaryotic translation initiation and of Biol. 11: PubMed Scopus Google Scholar). direct of translation initiation factors and the facilitate initiation of translation by mechanisms that not involve In the of the to the translational in of translation and of functions for translation and RNA PubMed Scopus Google Scholar). cellular mRNAs also can that may with the ISR. the amino acid mRNA to with embedded of the and during p-eIF2α and stress is to the to translation of the I. H. M. et of translational a upstream is the that of mRNA 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, I. C. H. et regulates translation in a to Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). in the RNA structures can enhance translation are of translated uORFs that may enhance or translation An important is to to translation efficiencies of mRNAs in the ISR 5′-leader In uORFs are to critical initiation as which enhance translation reinitiation Translational control by of eukaryotic 2016; PubMed Scopus Google Scholar, In that to ribosomes and upstream ORFs to Google Scholar, L. et with 5' of uORF1 to of scanning by ribosomes for reinitiation PubMed Scopus Google Scholar). as for the ISR uORFs can also codons that are to that to reinitiation L.R. T.D. Wek R.C. of eIF2 of upstream to enhance Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Palam L.R. C. Wek R.C. translation in the integrated stress Biol. 2016; Full Text Full Text PDF PubMed Scopus Google In the of the translated uORF2 encodes to the This is to translation reinitiation at the downstream Willy J.A. C. Wek R.C. reinitiation translation and regulates the integrated stress Biol. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). ATF4 and the mRNAs uORF2 that with the which recognition of the start codons K.M. Wek R.C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.Proc. Natl. Acad. PubMed Scopus Google Scholar, Palam L.R. L. Wek R.C. of eIF2 translational control in response to diverse stress Biol. Full Text Full Text PDF PubMed Scopus Google Our study mechanism RNA stem-loop downstream of can together to reinitiation The stability of the in the IBTKα mRNA is which not scanning by the of stem-loop structures can as a to scanning M. and mechanisms of of translation by secondary in Biol. PubMed Scopus Google Scholar). with the downstream of uORF2 in the 5′-leader of the IBTKα stability is to be to for the to in with the translated uORF2 to serve as a in the IBTKα translational a the for IBTKα translational control that of the translational in the ISR not only translated uORFs also the and stability of RNA secondary human cells with in this from in with and and IBTKα and IBTKα cells from The used to in 2 of the IBTKα in a in the coding sequence. In this of human gene used to in a with The RNA in the a of IBTKα IBTKα and in and the efficiency by that used as a to by for The K. et of from PubMed Scopus Google used to the of in the IBTKα and IBTKα cells to control cells the control of by the expression the protein from control IBTKα IBTKα and cells and the of IBTKα protein by analyses as IBTKα and IBTKα cells in a and in to and with or of by of with and and from the a and protein in and with by at at with protein to the in protein with a of and of protein of each by in a of IBTKα used to protein used the to the IBTKα ATF4 Palam L.R. L. Wek R.C. of eIF2 translational control in response to diverse stress Biol. Full Text Full Text PDF PubMed Scopus Google eIF2 p-eIF2α The of IBTKα by from the from IBTKα cells with or and IBTKα cells in with targeted the secondary in combination with a IBTKα by and the of IBTKα the with as a A encoding the 5′-leader of the human IBTKα gene transcript the and the in the from The reporter cells and assays as the 5′-leader of the ATF4 mRNA the the and the The IBTKα and ATF4 used as to the in the and in the 5′-leader the encoding the or the of the by with from of a of and the for at the of the and the with The for by the open in the 2 cells The and of IBTKα with with the of each of cells used for for and used for RNA for of the reporter of cells with or 2 C. Zhang K. et of a with and PubMed Scopus Google or for by from the and cells with of to each well to for the at for to for each to by of the used for and a The and and the to for The to of to and as the The for each the by to to that of control and IBTKα cells in and the cells with or for to cells with and with and of polysome and to the with and in with with and by at for of the the of a to by at for 2 at of the gradient as T.D. Palam L.R. Fusakio M.E. Willy J.A. Davis C.M. McClintick J.N. et al.Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha.Mol. Biol. Cell. 2014; 25: 1686-1697Crossref PubMed Scopus (83) Google Scholar, T.D. Wek R.C. for eIF2 kinases and translational control in the protein PubMed Scopus Google a and a with used to the polysome A of from each which for a of the in and mRNA in the polysome in response to of control RNA each to for of of and RNA and as in the RNA and The gene transcript as T.D. Palam L.R. Fusakio M.E. Willy J.A. Davis C.M. McClintick J.N. et al.Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha.Mol. Biol. Cell. 2014; 25: 1686-1697Crossref PubMed Scopus (83) Google Scholar, T.D. Wek R.C. for eIF2 kinases and translational control in the protein PubMed Scopus Google Scholar). the in and mRNA as mRNA in during mRNA mRNA in during mRNA is that the human IBTKα mRNA encodes a ATF4 and transcripts are The ATF4 contains the of which is codons in translated IBTKα transcripts are in the of the The polysome to by the of the polysome The polysome is as the of the that contains the and the of IBTKα and IBTKα cells with or for and RNA to reporter RNA from or cells that with IBTKα or to the for each The of RNA for the by A of of RNA used for to with of used well for The by The for the are in the The for each transcript by with as in transcripts with by the multiple in the reporter assays the and at by a used to multiple are the and and are for This contains and in a C. is a of the of The also to and for and of Wek for H. and C. H. and C. H. K. and C. C. H. K. and C. H. C. and K. C. C. and H. C. C. H. and K. C. H. K. and C. H. K. and C. H. 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