The evolutionarily conserved checkpoint protein kinase, TOR (target of rapamycin), has emerged as a major effector of cell growth and proliferation via the regulation of protein synthesis. Work in the last decade clearly demonstrates that TOR controls protein synthesis through a stunning number of downstream targets. Some of the targets are phosphorylated directly by TOR, but many are phosphorylated indirectly. In this review, we summarize some recent developments in this fast-evolving field. We describe both the upstream components of the signaling pathway(s) that activates mammalian TOR (mTOR) and the downstream targets that affect protein synthesis. We also summarize the roles of mTOR in the control of cell growth and proliferation, as well as its relevance to cancer and synaptic plasticity.

/pdf/upstream-and-downstream-of-mtor-2v5cvgfhmb.pdf

Upstream and downstream of mTOR

Stress granules: the Tao of RNA triage

The most abundant mRNA post-transcriptional modification is N(6)-methyladenosine (m(6)A), which has broad roles in RNA biology. In mammalian cells, the asymmetric distribution of m(6)A along mRNAs results in relatively less methylation in the 5' untranslated region (5'UTR) compared to other regions. However, whether and how 5'UTR methylation is regulated is poorly understood. Despite the crucial role of the 5'UTR in translation initiation, very little is known about whether m(6)A modification influences mRNA translation. Here we show that in response to heat shock stress, certain adenosines within the 5'UTR of newly transcribed mRNAs are preferentially methylated. We find that the dynamic 5'UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well-characterized m(6)A 'reader'. Upon heat shock stress, the nuclear YTHDF2 preserves 5'UTR methylation of stress-induced transcripts by limiting the m(6)A 'eraser' FTO from demethylation. Remarkably, the increased 5'UTR methylation in the form of m(6)A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single m(6)A modification site in the 5'UTR enables translation initiation independent of the 5' end N(7)-methylguanosine cap. The elucidation of the dynamic features of 5'UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m(6)A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.

Dynamic m6A mRNA methylation directs translational control of heat shock response

In eukaryotes, the translation initiation codon is generally identified by the scanning mechanism, wherein every triplet in the messenger RNA leader is inspected for complementarity to the anticodon of methionyl initiator transfer RNA (Met-tRNAi). Binding of Met-tRNAi to the small (40S) ribosomal subunit, in a ternary complex (TC) with eIF2-GTP, is stimulated by eukaryotic initiation factor 1 (eIF1), eIF1A, eIF3, and eIF5, and the resulting preinitiation complex (PIC) joins the 5' end of mRNA preactivated by eIF4F and poly(A)-binding protein. RNA helicases remove secondary structures that impede ribosome attachment and subsequent scanning. Hydrolysis of eIF2-bound GTP is stimulated by eIF5 in the scanning PIC, but completion of the reaction is impeded at non-AUG triplets. Although eIF1 and eIF1A promote scanning, eIF1 and possibly the C-terminal tail of eIF1A must be displaced from the P decoding site to permit base-pairing between Met-tRNAi and the AUG codon, as well as to allow subsequent phosphate release from eIF2-GDP. A second GTPase, eIF5B, catalyzes the joining of the 60S subunit to produce an 80S initiation complex that is competent for elongation.

The Scanning Mechanism of Eukaryotic Translation Initiation

The eukaryotic translation initiation factor 4B (eIF4B) plays a critical role in recruiting the 40S ribosomal subunit to the mRNA. In response to insulin, eIF4B is phosphorylated on Ser422 by S6K in a rapamycin-sensitive manner. Here we demonstrate that the p90 ribosomal protein S6 kinase (RSK) phosphorylates eIF4B on the same residue. The relative contribution of the RSK and S6K modules to the phosphorylation of eIF4B is growth factor-dependent, and the two phosphorylation events exhibit very different kinetics. The S6K and RSK proteins are members of the AGC protein kinase family, and require PDK1 phosphorylation for activation. Consistent with this requirement, phosphorylation of eIF4B Ser422 is abrogated in PDK1 null embryonic stem cells. Phosphorylation of eIF4B on Ser422 by RSK and S6K is physiologically significant, as it increases the interaction of eIF4B with the eukaryotic translation initiation factor 3.

/pdf/the-mtor-pi3k-and-mapk-pathways-converge-on-eif4b-to-control-4k0rcgeqd7.pdf

The mTOR/PI3K and MAPK pathways converge on eIF4B to control its phosphorylation and activity

http://www.genebee.msu.su/LPSR/Rubtsova_JBC2003.pdf

Distinctive Properties of the 5′-Untranslated Region of Human Hsp70 mRNA

The reconstitution of translation initiation complexes from purified components is a reliable approach to determine the complete set of essential canonical initiation factors and auxiliary proteins required for the 40S ribosomal subunit to locate the initiation codon on individual mRNAs. Until now, it has been successful mostly for formation of 48S translation initiation complexes with viral IRES elements. Among cap-dependent mRNAs, only globin mRNAs and transcripts with artificial 5' leaders were amenable to this assembly. Here, with modified conditions for the reconstitution, 48S complexes have been successfully assembled with the 5' UTR of beta-actin mRNA (84 nucleotides) and the tripartite leader of adenovirus RNAs (232 nucleotides), though the latter has been able to use only the scanning rather then the shunting model of translation initiation with canonical initiation factors. We show that initiation factor 4B is essential for mRNAs that have even a rather moderate base pairing within their 5' UTRs (with the cumulative stability of the secondary structure within the entire 5' UTR < -13 kcal/mol) and not essential for beta-globin mRNA. A recombinant eIF4B poorly substitutes for the native factor. The 5' UTRs with base-paired G residues reveal a very sharp dependence on the eIF4B concentration to form the 48S complex. The data suggest that even small variations in concentration or activity of eIF4B in mammalian cells may differentially affect the translation of different classes of cap-dependent cellular mRNAs.

http://www.genebee.msu.ru/LPSR/Dmitriev_MCB2003.pdf

Assembly of 48S Translation Initiation Complexes from Purified Components with mRNAs That Have Some Base Pairing within Their 5′ Untranslated Regions

Rhopalosiphum padi virus (RhPV) is an insect virus of the Dicistroviridae family. Recently, the 579-nucleotidelong 5 untranslated region (UTR) of RhPV has been shown to contain an internal ribosome entry site (IRES) that functions efficiently in mammalian, plant, and insect in vitro translation systems. Here, the mechanism of action of the RhPV IRES has been characterized by reconstitution of mammalian 48S initiation complexes on the IRES from purified components combined with the toeprint assay. There is an absolute requirement for the initiation factors eIF2 and eIF3 and the scanning factor eIF1 to form 48S complexes on the IRES. In addition, eIF1A, eIF4F (or the C-terminal fragment of eIF4G), and eIF4A strongly stimulated the assembly of this complex, whereas eIF4B had no effect. Although the eIF4-dependent pathway is dominant in the RhPV IRES-directed cell-free translation, omission of either eIF4G or eIF4A or both still allowed the assembly of 48S complexes from purified components with 23% of maximum efficiency. Deletions of up to 100 nucleotides throughout the 5-UTR sequence produced at most a marginal effect on the IRES activity, suggesting the absence of specific binding sites for initiation factors. Only deletion of the U-rich unstructured 380-nucleotide region proximal to the initiation codon resulted in a complete loss of the IRES activity. We suggest that the single-stranded nature of the RhPV IRES accounts for its strong but less selective potential to bind key mRNA recruiting components of the translation initiation apparatus from diverse origins.

http://www.genebee.msu.ru/LPSR/Terenin_MCB2005.pdf

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry.

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus

http://www.genebee.msu.ru/LPSR/Andreev_MCB2006.pdf

Sergei E. Dmitriev

Papers

Distinctive Properties of the 5′-Untranslated Region of Human Hsp70 mRNA

Assembly of 48S Translation Initiation Complexes from Purified Components with mRNAs That Have Some Base Pairing within Their 5′ Untranslated Regions

A cross-kingdom internal ribosome entry site reveals a simplified mode of internal ribosome entry.

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors.