UORFs-mediated Translational Regulation Of γ-globin And PD-L1

β-thalassemia is one of the most common hematological diseases caused by genetic mutations at HBB gene(coding β-globin protein)on Chromosome 11.Currently,pharmaceutical induction of the fetal version of hemoglobin protein,y-globin,to compensate for the loss of β-globin protein is one of the therapeutic strategies of treating β-thalassemia.Therefore,to fully understand the mechanism by which the expression of y-globin is regulated remians crucial.Recent reports have shown that y-globin gene is epigenetically regulated by DNA or histone methylations,while a major member of Type Ⅱ Protein Arginine Methyltransferases,PRMT5,has been shown to regulate y-globin transcription through its epigenetic modifications of the histone H4 at the globin gene promoter.However,whether other PRMT family members,especially PRMT1,a member of Type Ⅰ methyltransferase family can modify the same histone site(H4R3)as PRMT5,and regulate the expression of y-globin remains unclear.Here we first utilized a new inhibitor of Type Ⅰ PRMTs,PT1601,to test the regulation of PRMT1 on y-globin.We found that PT1601 can efficiently induce the expression of γ-globin mRNA in both K562 and HUDEP-2 cells.Interestingly,this effect is not due to the inhibition of the emzyme activity of PRMT 1 by PT1601.Instead,it might be due to the increase in PRMT1PRMT5 interaction,which suppresses the functional PRMT5 homodimer binding to γ-globin promoter to derepress γ-globin gene transcription.Suprisingly,either a deletion mutation of the catalytic domain of PRMT 1 or its knockdown results in an upregulation of the abundance of γglobin protein instead of its mRNA,indicating that PRMT1 may be involved in the posttranscriptional regulation,e.g.the translational regulation of y-globin mRNA,of which the mechanism remains largely unknown.In order to uncover the mechanism underlying the regulation of y-globin mRNA translation,as well as the role of PRMT1 in this process,we first developed luciferase reporter and polysome fractionation assays,and proved that the loss of enzymatic function of PRMT1 can significantly upregulate the translation of y-globin mRNA.We show that induction of the endoplasmic reticulum(ER)stress by Tunicamycin results in an increase in y-globin protein abundance rather than its mRNA level.Together with y-globin 5’UTR reporter assay showing that its mRNA translation is regulated by its 5’UTR,we proposed that upstream open reading frame(uORF)may play a crucial role in this regulation.After a careful analysis of the y-globin 5’UTR,we mutated the putative uORFs,and identified that a non-classical upstream initiation codon "AUC",as a barrier of downstream y-globin translation.We found that mutating AUC can promote the bypass of this uORF,and initiate the translation from downstream canonical AUG start codon of the main ORF by the ribosome.In addition,we observed a profund decreasse of γ-globin 5’UTR reporter activity when we mutate AUC to a stronger start codon AUG.These results suggest that the uprstream AUC sequence is the start codon of the putative uORF in γ-globin 5’UTR,which may suppresse the translation of downstream y-globin ORF.Furthermore,we found that induction of ER stress or mutation of AUC initation codon under the condition of PRMT1 catalytic activity loss did not further increase the reporter activity of γ-globin 5’UTR,indicating that PRMT1 is indeed involved in the uORF-mediated translation repression of y-globin.Lastly,we found that,besides its function in regulating y-globin translation,PRMT1 may also control the translation of c-MYC,a key regulator of hematopoitic stem cells(HSCs)differentian and self-renewal which may function in K562 cell differention.In summary,this project not only demonstrates the important role of translation control in hematopoiesis and globin gene expression,but also highlights that targeting the components of translation machinery(such as PRMT1)may serve as a putative strategy of treating β-thalassemia.Recently,there is an increased realization of the fact that translation control is not only a crucial determinant of normal cell fate,but also a network that tumor cells are widely hijacking to promote the expression of cancer-promoting proteins.This is usually rendered by the cooperation of various oncogenes.To understand how oncogenes synergize the translation of pro-tumor mRNAs,our preliminary work generated a genetic mouse model with the co-activation of two most common oncogenes,c-MYC and KRAS(MYCTg;KRASG12D).This preliminary work shows that the two oncogene synergize to promote a more aggressive tumor,with a more malignant primary tumor growth and a metastasis to the lung,compared to the activation of a single oncogene KRAS.Intriguingly,genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared to KRASG12D revealed potential alterations in translation of mRNAs,including programmed deathligand 1(PD-L1).We first confirmed that PD-L1 is indeed translationally regulatied by c-MYC and KRAS cooperation,which is not controlled by potential motifs within PD-L1 5’UTR.Further analyses revealed that PD-L1 translation is repressed in KRASG12D tumors by functional,non-canonical uORFs within its 5’UTR,which is bypassed in MYCTg;KRASG12D tumors,at least partially through the induction of eIF2α phosphorylation,to evade immune attack and develop metastasis.Furthermore,with CRISPR/Cas9 guided genome editing of these uORF start codons,KRASG12D tumors encountered profund increases in PD-L1 protein abundance and the incidence of metastasis formation.Lastly,we also discovered that c-MYC and KRAS cooperation can also induce the translation of factors such as cell cycle mRNAs,CDC20 and PLK1,as well as cytokines that can recruit immune cells,CCL2 and CXCL9,potentially through bypassing the uORFs within their 5’UTRs.Together,these studies reveal how immune-checkpoint and other pro-tumor proteins can be manipulated by distinct oncogenes at the level of mRNA translation,which can be exploited for new immunotherapies.In summary,translation regulation has a widespread and profound influence on various biological processes.The two projects above not only reveal new paradigms of uORF-mediated translational inhibition,but also comprehensively illustrate the significance of translational regulation in development and disease,which may contribute to the development of new therapeutic targets and treatment.