Cap and cap‐binding proteins in the control of gene expression

Diagrams representing the similarities in molecular architecture of the cap‐binding pockets of eIF4E, CBC20, and VP39. In each protein, aromatic residues that intercalate the cap are shown in red, residues binding the functional groups of the guanine base are shown in green, residues interacting with the 5′‐5′ triphosphate bridge are shown in purple, and residues that stabilize the 7‐methyl group are shown in blue. Cap analogs are shown in white. Diagrams were generated using PyMOL software (http://www.pymol.org). eIF4E‐PDB accession number 1L8B; CBP 20‐PDB accession number 1H2T; VP39‐PDB accession number 1AV6.

Emerging mechanisms of the regulation of capping. (a) Efficiency of cap methylation is increased by stimulation of RNA (guanine‐N7‐) methyltransferase (MT) activity by ubiquitin‐conjugase Cdc34 in yeast, Xenopus S‐adenosyl homocysteine hydrolase (xSAHH) which eliminates the inhibitory by‐product of cap methylation, S‐adenosyl homocysteine (SAH), by interaction with importin α (Impα), which is inhibited by importin β (Impβ), and by c‐myc and E2F1 transcription factors. It was suggested that c‐myc stimulates transcription of SAHH. Abbreviations: SAM, S‐adenosyl methionine. (b) Upon decapping, most mRNAs are rapidly degraded by 5′ → 3′ exonucleases. Several transcripts, including β globin mRNA from the erythroid cells of β thalassemia patients has been shown to produce stable degradation products. It is thought that these fragments are capped by 140 KDa cytoplasmic capping complex that exhibits polynucleotide 5′‐monophosphate RNA kinase (PMRK), guanylyltransferase (GT) and RNA (guanine‐N7‐) methyltransferase (MT) activities.

Viruses developed mechanisms to utilize cap structure to ensure efficient expression of their genes. (a) Vaccinia virus viral protein 39 (VP39) binds the cap and functions as 2′‐O‐ribose methyltransferase, catalyzing the transfer of a methyl group to the first transcribed nucleotide (N1) of mRNA and converting cap 0 to cap 1. (b) RNA‐dependent RNA polymerase of vesicular stomatitis virus (VSV), viral protein L, forms the cap by a mechanism distinct from cellular capping enzymes and VP39, adding GDP to monophosphate ends before 2′‐O and then m7G methylation. (c) Flu virus RNA polymerase utilizes a ‘cap‐snatching’ mechanism to prime synthesis of flu mRNA with host capped mRNA fragments. (d) Cytomegalovirus (CMV) developed strategies to overtake the host's translational machinery by stabilizing the eIF4F complex (via pUL69) and by activating the mTORC1 pathway (via pUL38). (e) N protein of hantavirus appears to act as a substitute for eIF4F activity in the cell. Abbreviations: eIF4E‐binding proteins (4E‐BPs); mammalian target of rapamycin complex 1 (mTORC1); PABP, poly(A) binding protein; eIF, eukaryotic translation initiation factor.

The regulation of eIF4E activity. The activity of eIF4E is regulated by proteins that compete for binding to its dorsal surface (residues V69‐W73 in mammalian eIF4E). There is an allosteric communication between dorsal surface and cap binding pocket which are on opposite sides of the eIF4E molecule (red arrow). Proteins that bind to dorsal surface either stimulate (eIF4G) or inhibit [eIF4E‐binding proteins (4E‐BPs), Cup and Maskin] translation. Although 4E‐BPs inhibit translation of bulk cellular mRNAs, they preferentially affect ‘eIF4E‐sensitive’ mRNAs which harbor long and structured 5′ UTRs. Activity of 4E‐BPs in the cell is regulated by the mammalian target of rapamycin complex 1 (mTORC1) pathway. In contrast, Maskin and Cup inhibit translation of a limited number of specific mRNAs, where specificity is determined by the presence of the cytoplasmic polyadenylation element (CPE) or bruno response element (BRE) in the mRNA 3′ UTR, respectively. In addition, several homeobox proteins have been shown to interact with the dorsal surface of eIF4E family memebrs. eIF4E activity is also regulated via phosphorylation at residue S209 by the MAP kinase integrating kinases 1 and 2 (Mnk1 and 2). The effects of eIF4E phosphorylation on its translational activity and cap binding are still unclear. eIF4E is a transcriptional target of c‐myc, whereas c‐myc is regulated by eIF4E at the level of translation. Abbreviations: CPEB, cytoplasmic polyadenylation element binding protein; p38MAPK, mitogen‐activated protein kinase; Erk1/2, extracellular signal‐regulated kinase 1 and 2; AMPK, adenine monophosphate‐activated protein kinase; LKB1, serine/threonine kinase 11; REDD1, DNA‐damage‐inducible transcript 4; PI3K, Phosphoinositide 3‐kinase; Rags, ras‐related GTP‐binding proteins; eIF, eukaryotic translation initiation factor.

Schematic representation of cap‐dependent processes governed by cap‐binding proteins. Capping occurs co‐transcriptionally where capping enzyme (CE) consisting of the RNA triphosphatase and guanylyltransferase and RNA (guanine‐N7‐) methyltransferase (MT) are recruited to nascent transcripts through interaction with the TFIIH‐phosphorylated C‐terminal domain (CTD) of polymerase II (Pol II). After the cap formation, nuclear cap binding complex (nCBC) consisting of cap‐binding proteins CBP 20 and 80 binds the mRNA and with other protein complexes mediates its effects on the subsequent steps of mRNA metabolism. After mRNA is exported from the nucleus, eIF4E binds the cap and recruits it to the small ribosomal subunit. In addition, eIF4E was suggested to export a subset of mRNAs from the nucleus and to play a role in Staufen‐mediated decay (SMD). Finally, cap is removed by decapping enzyme (Dcp1 and 2), after which mRNA is rapidly degraded. Abbreviations: TFIIH, transcription factor II H; NXF1, nuclear export factor 1; REF, RNA and export factor binding protein; TREX, transcription/export complex; EJC, exon junction complex; PABP, poly(A) binding protein; eIF, eukaryotic translation initiation factor; 43S, 43S pre‐initiation complex; Dcp, decapping protein complex.