Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes

Abstract

cells subjected to environmental stress, untranslated mRNA accumulates in discrete cytoplasmic foci that have been termed stress granules. Recent studies have shown that in addition to mRNA, stress granules also contain 40S ribosomal subunits and various translation initiation factors, including the mRNA binding proteins eIF4E and eIF4G. However, eIF2, the protein that transfers initiator methionyl-tRNA i (Met-tRNAi) to the 40S ribosomal subunit, has not been detected in stress granules. This result is surprising because the eIF2 ⅐ GTP ⅐ Met-tRNA i complex is thought to bind to the 40S ribosomal subunit before the eIF4G ⅐ eIF4E ⅐ mRNA complex. In the present study, we show in both NIH-3T3 cells and mouse embryo fibroblasts that stress granules contain not only eIF2 but also the guanine nucleotide exchange factor for eIF2, eIF2B. Moreover, we show that phosphorylation of the ␣-subunit of eIF2 is necessary and sufficient for stress granule formation during the unfolded protein response. Finally, we also show that stress granules contain many, if not all, of the components of the 48S preinitiation complex, but not 60S ribosomal subunits, suggesting that they represent stalled translation initiation complexes. eIF4E; eIF4G; eIF3; unfolded protein response; PERK ONE OF THE BEST-CHARACTERIZED mechanisms for regulating mRNA translation in eukaryotic cells involves phosphorylation of the ␣-subunit of eukaryotic initiation factor, eIF2 (reviewed in Ref. 16). During initiation, eIF2 forms a complex with GTP and initiator methionyl-tRNA i (met-tRNA i ), and this ternary complex subsequently binds to the 40S ribosomal subunit to form the 43S preinitiation complex (reviewed in Refs. 10 and 24). Through the action of a translation initiation factor complex referred to as eIF4F, which is comprised of eIF4A, eIF4E, and eIF4G, mRNA is bound to the 43S preinitiation complex, resulting in formation of the 48S preinitiation complex (reviewed in Ref. 20). During a later step in initiation, the GDP bound to eIF2 is hydrolyzed in an eIF5-mediated process and initiation factors are released from the ribosome. Before binding Met-tRNA i and reforming the ternary complex, the GDP bound to eIF2 must be exchanged for GTP, a reaction that is catalyzed by the guanine nucleotide exchange factor, eIF2B. One mechanism for regulating the activity of eIF2B involves phosphorylation of the ␣-subunit of eIF2 on Ser51, an event that converts eIF2 from a substrate into a competitive inhibitor of eIF2B (reviewed in Ref. 10). Thus, by inhibiting eIF2B, phosphorylation of eIF2␣ results in a global inhibition of protein synthesis.Hyperphosphorylation of eIF2␣ occurs under a variety of conditions that result in disruption of normal cell homeostasis. For example, conditions that impede correct folding of newly synthesized proteins in the lumen of the endoplasmic reticulum (ER), i.e., the so-called unfolded protein response, result in activation of the eIF2␣ kinase, PERK (also referred to as PEK) (28). PERK is a trans-ER membrane protein with a luminal domain...

Arabidopsis CML38, a Calcium Sensor That Localizes to Ribonucleoprotein Complexes under Hypoxia Stress

“…SG particles consist of mRNA bound in an arrested state as a preinitiation (48S) complex composed of small 40S ribosomal subunits, eIFs, and mRNA-associated proteins like the poly(A)-binding protein (Kedersha et al, 2002;Kimball et al, 2003). Besides these core components, proteomic analyses of SGs reveal that these structures are complex, with a large array of protein components, including RNAprocessing proteins, metabolic enzymes, chaperone proteins, and cell signaling components, associated with the granule complex (Samaha et al, 2010;Isabelle et al, 2012;Sorenson and Bailey-Serres, 2014).…”

“…Similar to previous studies of time-resolved SG disassembly in plant tissues recovering from hypoxia (Sorenson and BaileySerres, 2014) and related heat stress (Gutierrez-Beltran et al, 2015), the CML38 foci disassemble upon reoxygenation. Second, MS analysis of immunoprecipitates of CML38 from hypoxic root samples reveals the presence of established SG-associated proteins, including poly(A)-binding protein (Kedersha et al, 2002;Kimball et al, 2003;Sorenson and Bailey-Serres, 2014), eukaryotic initiation factors (Li et al, 2010), eukaryotic elongation factors (Hafrén et al, 2013), RNA helicase (Koroleva et al, 2009), small ribosomal subunit proteins (Kedersha et al, 2002;Sorenson and Bailey-Serres, 2014), and others (Mazroui et al, 2007;Buchan et al, 2013;Yan et al, 2014, GutierrezBeltran et al, 2015. Third, coexpression of the core SG-nucleating RNA-binding protein RBP47 (Weber et al, 2008) with CML38 shows that, under hypoxia conditions, the two proteins colocalize to cytosolic granules.…”

Assessment of biomarkers of protein anabolism in skeletal muscle during the life span of the rat: sarcopenia despite elevated protein synthesis

“…Phosphorylation of eIF2␣ on Ser 51 (25), eIF2B⑀ on Ser 535 (36), and S6K1 on Thr 389 (1) was measured by Western blot analysis, as described previously, using antiphosphopeptide antibodies that specifically recognize eIF2␣ on Ser 51 (BioSource International), eIF2B⑀ on Ser 535 (BioSource International), or S6K1 phosphorylated at Thr 389 (New England Biolabs, Beverly, MA).…”

A genome-wide RNAi screen identifies genes regulating the formation of P bodies in C. elegans and their functions in NMD and RNAi

“…SGs are physically distinct and spatially separable from P bodies, playing a role in storage of translationally repressed mRNAs under stress conditions (Kimball et al, 2003;Kedersha et al, 2005). The assembly of SGs appears to be regulated by distinct signaling pathways in mammalian cells.…”