Research

Figure 1. Model of cap-dependent translation initiation (m7G)*. This mechanism predominates in eukaryotic cells (85-90% of translation initiation) and is activated by the mTOR kinase complex 1 (mTORC1), which phosphorylates the p70S6K kinase (S2) and the 4E-BP protein (S1), whose phosphorylation enables dimerization and activation of the eukaryotic translation initiation factor (eIF4E). Activation of the p70S6K kinase leads to the phosphorylation of the S6 protein of the small ribosomal subunit (rpS6) and translation initiation factors: eIF3 and eIF4B/eIF4A. p70S6K also participates in the regulation of eIF4G, as well as eEF1A and eEF2K/eEF2. The initiation mechanism includes the sequential association of fundamental protein complexes (marked as C. in orange text): a) the ternary complex eIF2◦GTP◦Met-tRNAiMet (TC), containing the translation initiation factor eIF2 with an unphosphorylated alpha subunit, guanosine triphosphate (GTP), and initiator aminoacyl-tRNA (Met-tRNAiMet); b) the multifactorial protein complex MCF (MCF complex), assembled on the small 40S ribosomal subunit, containing various translation initiation factors such as eIF1, eIF1A, eIF3, and eIF5; c) the eIF4F factor complex (eIF4F complex), formed around the eIF4G factor, which serves as a scaffold for the eIF4E factor binding to the 5’ cap, eIF4B, eIF4H, and the eIF4A helicase. The eIF4G factor also binds other proteins (outside the eIF4F complex), such as the poly(A)-binding protein (PABP), the Mnk1 kinase, and the eIF3 factor. The three complexes mentioned (highlighted in beige) organize into more complex structures: the 43S preinitiation complex (PIC 43S), in which the eIF2 factor connects the TC complex with the MCF complex and the small ribosomal subunit, as well as the 48S preinitiation complex (PIC 48S), where the eIF3 factor links the PIC (43S) complex with the eIF4G factor of the eIF4F complex. The eIF2 factor also ensures the correct positioning of the initiating aminoacyl-tRNA in the P site of the ribosomal subunit, which contains three tRNA-binding sites (A, P, and E). The encounter of the PIC complex with the start codon (AUG) ends the scanning process of the 5’UTR and initiates the release phase of the initiation factors. This stage includes sequentially: eIF5-dependent hydrolysis of GTP bound to eIF2 (ternary complex TC), binding and hydrolysis of GTP by eIF5B, dissociation of eIF1, eIF2-GDP, eIF5, followed by eIF1A and eIF5B-GDP, binding of the large 60S ribosomal subunit, formation of the elongation complex (80S), and the beginning of synthesis based on the coding sequence (CDS) in the correct reading frame (ORF). The regeneration of TC complex components occurs through the exchange of GDP for GTP in eIF2, catalyzed by the eIF2B factor. The diagram highlights inhibition sites (H) of cap-dependent translation initiation (red stars), including: phosphorylation of eIF2α(Ser51) (H3), as well as inhibition of phosphorylation of mTOR effector proteins such as 4E-BP proteins, which in their non-phosphorylated form block eIF4E at the eIF4G binding site (H1), and the p70S6K kinase, which in its non-phosphorylated form associates with eIF3 (H2). mTOR phosphorylation is inhibited by hypoxia and inhibitors such as rapamycin. The RBM4 protein inhibits cap-dependent translation while simultaneously enhancing synthesis initiated by IRES and rHRE elements. ISRIB - an integrated stress response (ISR) inhibitor that restores cap-dependent translation.

*Reference: Master A, Nauman A. Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE). Postepy Biochem. 2014;60(1):39-54. Polish. PMID: 25033541. 

Figure 2. General model of IRES-dependent translation initiation (cap-independent translation). The protein requirements and mechanisms of IRES-initiated translation can vary significantly between transcripts of different genes, as a result of different IRES sequences in the 5’UTR as well as secondary and tertiary spatial structures, schematically indicated in the figure with the IRES label. However, most IRES elements can be stimulated by IRES-specific trans-acting factors (ITAFs), which include ribonucleoproteins. The general model of IRES-dependent initiation assumes: a) no requirement for the eIF4E factor (which binds the 5’ cap) to be attached to the preinitiation complex, as its inhibition (e.g., by 4E-BP protein) typically does not suppress IRES-dependent translation (red star); b) no requirement for the removal of the phosphate residue (P in a circle) from the Ser⁵¹ residue of the eIF2α subunit, whose phosphorylation (e.g., under cellular stress conditions) inhibits cap-dependent translation initiation but not IRES-dependent initiation (red star). This phosphorylation leads to the inhibition of the activity of the eIF2◦GTP◦Met-tRNAiMet ternary complex (TC complex) by preventing its reuse, which depends on the eIF2B-mediated exchange of GDP for GTP. In the general model, IRES-initiated translation also does not require the presence of the entire eIF4G protein but may require RNA looping (circularization), facilitated by interactions between poly(A)-binding proteins (PABP) and eIF4G, which increases the frequency of translation reinitiation. The requirements for other initiation factors may vary between individual IRES elements, ranging from none to all essential translation initiation factors, including the ternary complex TC (TC complex), the multifactorial MCF complex (MCF complex), and the eIF4F factor complex (eIF4F complex). Among classical initiation factors, IRES sequences most commonly require the presence of eIF4A and eIF3. Other explanations are provided in the main text. 

*Reference: Master A, Nauman A. Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE). Postepy Biochem. 2014;60(1):39-54. Polish. PMID: 25033541. 

Figure 3. Minimal model of IRES-dependent translation initiation. The initiation of translation in a small group of identified cellular IRES elements does not require any factors (interacting exclusively with ribosomal proteins) or has minimal requirements in this regard. However, most IRES elements can be activated by IRES-specific trans-acting factors (ITAFs), which include a heterogeneous group of nucleoproteins. As recently demonstrated, small non-coding microRNAs can also act as repressive ITAF factors. The IRES-B in the 5’UTR of the VEGF-A transcript can be specifically inhibited by miR-16. The assumptions of the minimal model largely overlap with the general model of IRES-dependent translation initiation (Fig. 2). However, it also suggests that the function of eIF2 (a.), which involves delivering the special initiator aminoacyl-tRNA Met-tRNAiMet (position P of the small ribosomal subunit), can also be taken over by other proteins such as eIF5B (b.), DENR (c.), MCT-1 (d.), Ligatin (e.), and many others (f.), which may explain the insensitivity of IRES systems to phosphorylation of the eIF2α subunit. IRES-dependent translation can be initiated by both the main start codon (AUG) and an alternative codon (CUG) located upstream (toward the 5’ end) of the AUG codon, allowing translation in the correct or an alternative reading frame (uORF). The VEGF-A transcript can encode both the normal protein, whose translation is initiated from IRES-A, as well as an elongated (more active) L-VEGF-A protein, synthesized from a second IRES-B located upstream, near the alternative start codon CUG.

*Reference: Master A, Nauman A. Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE). Postepy Biochem. 2014;60(1):39-54. Polish. PMID: 25033541. 

Figure 4. Model of rHRE-dependent translation initiation. This mechanism is cap-dependent and is initiated under hypoxic conditions, when phosphorylation of the eIF2α subunit (red star) occurs in response to stress, leading to the inhibition of classical cap-dependent translation. Protein synthesis initiation includes the following steps: a) binding of the RNA-binding adaptor protein RBM4 to the rHRE element in the 3’UTR, accompanied by b) the binding of the hypoxia-inducible factor HIF-2α by RBM4, c) the recruitment of eIF4E2 (a homolog of eIF4E) by the HIF-2α–RBM4 complex, allowing eIF4E2 to bind the 5’ cap (m⁷G), and d) the formation of a minimal initiation complex: 5’ cap – eIF4E2 – HIF-2α – RBM4 – rHRE, which closes the mRNA loop and facilitates multiple rounds of translation reinitiation, involving other essential factors, including the helicase eIF4A, which strongly interacts with HIF-2α. Question marks indicate interactions between factors that have not yet been clearly defined in this translation model. Other explanations are provided in the main text.

*Reference: Master A, Nauman A. Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE). Postepy Biochem. 2014;60(1):39-54. Polish. PMID: 25033541. 

Figure 5. Model of translation activation and initiation control by Maskin.
A. Inhibition of translation initiation by Maskin involves its substitution in place of the eukaryotic translation initiation factor eIF4G, which blocks the organization of the translation initiation complex, eIF4F (beige highlight). eIF4G can competitively compete with Maskin for access to the binding site of the eIF4E factor, which recognizes the 5' cap (m7G) at the 5' end of mRNA. A specific poly(A) ribonuclease, PARN, also participates in repression, preventing the elongation of the poly(A) tail. B. Translation initiation is activated by the phosphorylation of the CPEB protein as well as Maskin itself, leading to: reorganization of factors, binding of the CPSF protein recognizing the polyadenylation signal (AAUAAA), and activation of the poly(A) polymerase, Gld2. Simultaneously, Maskin releases the interaction site of the eIF4E factor with eIF4G, enabling the formation of the full eIF4F complex initiating the translation process (ribosome – 60S and 40S subunits). Subsequently, PABP proteins attach to the poly(A) tail, and mRNA looping occurs, increasing translation efficiency by facilitating the rebinding of ribosomes. The Maskin complex may also include the helicase eIF4A3. Further explanations are provided in the text.

*Reference: Master A, Nauman A. Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE). Postepy Biochem. 2014;60(1):39-54. Polish. PMID: 25033541. 

Figure 6. Model of switching between 5' cap-dependent and uORF-dependent translation initiation mechanisms. Alternative mechanisms: 5' cap-dependent translation initiation (A) and uORF-dependent initiation (C), are switched by kinases activated under cellular stress (B), which phosphorylate eIF2α (the switch), simplified here by analogy to ski jumping. A. Under optimal conditions (without cellular stress), efficient, classical 5' cap-dependent translation occurs in the cytoplasm, characterized by rapid scanning of the 5'UTR (V↑) from uORF-1 (starting bar) to uORF-2 (ski jump threshold). Excessive speed of the translation machinery (the ski jumper), containing the ribosome and other translation initiation factors (including eIF2α), causes entry into an incorrect reading frame (ski jump threshold) and ejection of this machinery from the uORF-2 element (from the threshold) beyond the correct reading frame (ORF, located just below the threshold), preventing the synthesis of the transcription factor ATF4 protein (blockade). B. Conditions inducing cellular stress, such as hypoxia (O2↓), low glucose levels (Gluc.↓), amino acids (A.K↓), heme (Hem↓), or the presence of double-stranded, usually viral RNA (dsRNA↑). These conditions activate specific eIF2α kinases (B), such as PERK, GCN2, HRI, and PKR, which phosphorylate (P) serine 51 (Ser51-◌) of the eIF2α subunit, thereby blocking the classical 5' cap-dependent translation initiation mechanism and enabling the activation of alternative mechanisms dependent on the uORF element (C) or the previously discussed rHRE and IRES elements. Trans-ISRIB (Integrated Stress Response Inhibitor — Bis-glycolamide) inhibits the integrated stress response (ISR) triggered by eIF2α phosphorylation. ISRIB increases the concentration of the TC complex, restores 5' cap-dependent translation, and inhibits ATF4 synthesis.
C. Cellular stress can activate the translation initiation mechanism dependent on the presence of alternative reading frames uORF (uORF-1, uORF-2), located upstream (toward the 5' end) of the correct reading frame (ORF). Phosphorylation of eIF2α (the switch) slows down 5'UTR scanning (V↓), so that the translation machinery (the ski jumper) cannot eject from the uORF-2 element and, by sliding down the sequence, reaches the correct start codon (located just below the ski jump threshold). The reduced frequency of reinitiation and limited availability of the TC complex (Fig. 1) allow the translation machinery (containing the small 40S ribosomal subunit) to bypass uORF-2 without assembling the full ribosome on this element. Stress conditions are associated with limited energy resources in cells (ATP availability), which correspond to the slowing of scanning speed and ribosome assembly on uORF-2. Bypassing uORF-2 increases the frequency of ribosome assembly (80S) at the start of the correct reading frame (ORF). Further elongation leads to the synthesis of the complete ATF4 protein. The efficiency of ATF4 production under cellular stress increases significantly, enabling the activation of further stages of the ATF4-dependent stress response. Additional explanations are provided in the text.

*Reference: Master A, Nauman A. Molecular mechanisms of protein biosynthesis initiation--biochemical and biomedical implications of a new model of translation enhanced by the RNA hypoxia response element (rHRE). Postepy Biochem. 2014;60(1):39-54. Polish. PMID: 25033541.