Uncoupling stress granule assembly and translation initiation inhibition.
Cytoplasmic stress granules (SGs) are specialized regulatory sites of mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SG occurs via two pathways; the eukaryotic initiation factor (eIF) 2alpha phosphorylation-dependent pathway mediated by stress and the eIF2alpha phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4A and eIF4G. In this study, we investigated the effects of targeting different translation initiation factors and steps in SG formation in HeLa cells. By depleting eIF2alpha, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAi(Met) ternary translation initiation complexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SG formation. In contrast, depletion of the cap-binding protein eIF4E or preventing its assembly into eIF4F results in modest SG formation. Intriguingly, interfering with the last step of translation initiation by blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SG assembly. Our study identifies translation initiation steps and factors involved in SG formation as well as those that can be targeted without induction of SGs.
Département de Biologie Médicale, Centre Hospitalier Universitaire de Québec/Centre De Recherche Hôpital Saint-François D'assise, Université Laval, Quebec, QC, Canada.
Sophie
S
Mokas
John R
JR
Mills
Cristina
C
Garreau
Marie-Josée
MJ
Fournier
Francis
F
Robert
Prabhat
P
Arya
Randal J
RJ
Kaufman
Jerry
J
Pelletier
Rachid
R
Mazroui
D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D010766:Phosphorylation; D039102:Poly(A)-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D011422:Propionates / Q000494:pharmacology; D011485:Protein Binding; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014162:Transfection
C111495:EIF4H protein, human; D015852:Eukaryotic Initiation Factor-2; D039561:Eukaryotic Initiation Factor-4E; D039642:Eukaryotic Initiation Factors; D039102:Poly(A)-Binding Proteins; D011422:Propionates; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D012358:RNA, Transfer, Met; C031799:eIF-4B; C002167:2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide; D006160:Guanosine Triphosphate