EIF2B Structure And Its Relationship With VWM

eIF2B (eukaryotic initiation factor 2B) plays a key role in mRNA translation and its control. eIF2B comprises five different subunits (α-ε); eIF2Bε,the largest subunit, contains the catalytic domain which mediates GDP/GTP on eIF2. eIF2Bεshows substantial sequence similarity to eIF2Bγ, with which it forms a catalytic subcomplex. This complex binds to the regulatory subcomplex containing eIF2Bαβandδforming the holocomplex. Mutations in eIF2B genes cause a neurological disorder termed vanishing white matter (VWM), which shows marked phenotypic variability. To help understand the etiology of VWM, we have studied the assembly of the eIF2B complex and the functional effects of VWM mutations. Our data show that specific conserved residues in the N-terminal regions of eIF2Bεand eIF2Bγare important in the interaction between these subunits and/or the regulatory subcomplex. This region shows homology to nucleotidyl transferases but residues conserved in such enzymes are not required for GDP/GTP-exchange. A second conserved region of elF2Bεandγcontaining repeats of isoleucines (or similar residues) is also involved in intersubunit interactions, Ile 385 playing an essential role. We have tested the effects of VWM mutations in eIF2Bβγδε. They have diverse effects upon eIF2B complex formation and exchange activity, which may help to explain the diverse VWM phenotypes. In particular, (i) several mutations (especially those in the catalytic domain) cause decreased activity; (ii) VWM mutations in the catalytic domain impair substrate binding; (iii) in contrast, the eIF2B [V73G] mutation actually displays enhanced activity; (iv) several mutations in eIF2B(β,γorδshow little or no alteration in complex formation or activity; and (v) the eIF2B 8mutants A391D and R483W, which each cause congenital disease, have very different effects (R483W greatly impairs complex formation and activity, while A391D affects neither). There is thus no evident correlation between the effects of mutations on in vitro function and disease severity. Some mutations may affect other (so far unknown) functions of eIF2B. These data extend our understanding of the architecture of eIF2B complexes and will be helpful in advancing understanding of this key factor and its role in VWM. mTOR (mammalian target of rapamycin) exists in cells in two types of complexes termed mTORCl and mTORC2 respectively.So far,the biological functions of mTORC2 are not fully understood, the known functions of mTORC2 include cytoskeleton rearrangement, cell survival and so on. mTORCl, as a central regulator in cells is involved in the regulation of different intracellular signaling events, such as protein synthesis, gene transcription, cell cycle progression, autophagy and ribosome biogenesis.Ribosome biogenesis is an important process during cell division and is evolutionarily conserved which requires a high degree of coordination among different cell signaling events.It is essential for cell to keep a suitable proliferation rate and stable cell size.All three types of DNA dependant RNA polymerases are invovled in the Synthesis of ribosome, Pol I is responsible for the transcription of 47S pre-ribosomal RNA; PolⅡtranscript ribosomal protein mRNA; PolⅢis responsible for the transcription of 5s rRNA.lt is already indisputable that TORCl regulates ribosome biogenesis,and the detailed mechanism underneath has been partially revealed in yeast.Studies in yeast shows TORCl is shuttling between the nucleus and the cytoplasm, and can directly bind to Pol I promoter, phosphorylate certain transcriptional factors, therefore activate the transcription of Pol I. Similarly, TORCl is also able to bind to yeast PolⅢpromoter and directly activate PolⅢ, Likewise, the expression of ribosomal proteins in yeast is regulated by TORCl in transcription level.AfterwardS,Similar studies were performed in mammalian system which indicates,to some extents, mTORCl regulates ribosome biogenesis in a similar way as in yeast,but the differences between these two species are also very obivious:mTORCl also shuttles between cytoplasm and nucleus, and directly binds to polⅢpromoter,futhur activates polⅢtranscription, mTORCl can also activate the transcription of pol I, but so far there is no evidence shows that mTORCl can directly bind to pol I promoter, phosphorylate and activate certain transcription factors of pol I.It has been show that mTORCl regulate the expression of mammalian ribosomal proteins in translation level rather than transcription level, and if mTORCl is involved in the regulation of rRNA processing is not clear yet. In this study, we found that, in vivo and in vitro,mTORCl can directly interact with a protein termed BOP1 which play an important role in the 32S rRNA processing, and a truncated mutation of BOP1 which lack the first 264 amino acids can still interact with mTORCl in vitro, But not in vivo, suggesting that the interaction between BOP1 and mTORCl requires correct subcellular localization of BOP1. In addition, we found PeBoW(PESl-BOPl-WDR12) complex defects(siRNA knocking down experiment/ over expressing dominant negative mutation of BOP 1) have feedback effects on mTORCl signaling pathway by means of increasing the phosphorylation of the mTORCl specific substrate S6K1 and its kinase activity, without affecting the phosphorylation of another well known mTORCl substrate called 4EBP1. The phenomenon that only S6K1 but not 4EBP1 is activated in this situation (Actinomycin D as well as rapamycin behave in a similar way) suggests that mTORCl can regulate different downstream substrates independently in some special situations. We Also found that there is a mTORCl substrate specific motif called TOS motif in BOP1, mutating this motif can dramatically attenuates the dominant negative effects of BOP1△on S6K1 activation, blocking of rRNA processing and cell cycle progression. In our case, we failed to see any activation of p38,which indicate the mechanism of how BOP1△activate S6K1 is different from DNA damage responses in which case S6K1 is activated by p38 MAPK.Last,we found that BOP1 is a phosphorylated protein, suggesting BOP1 can be regulated by some kinases in post-translational level. We found S126S127 is one of the phosphorylation sites, PKA and CKII can phosphorylate BOP1 in vitro. Our results suggest that mTORCl plays an important role in rRNA processing, The block of rRNA processing caused by PeBoW complex defects will feed back on mTORCl and specifically activate S6K1, the biological significance of this feedback effect is still elusive.