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mRNA cap binding proteins: essential factors for initiating translation
Cell, Vol. 40, 223-224,
February
1985, Copyright
0 1985 by MIT
mRNA Cap Binding Proteins: Essential Factors for Initiating Translation Aaron Roche Roche Nutley,
J. Shatkin Institute of Molecular Research Center New Jersey 07110
Biology
The presence of a 5’-terminal “cap:’ m7G(5’)ppp(5’)N, is a nearly ubiquitous feature of eukaryotic mRNAs. Capping is an early reaction, occurring at the 5’-triphosphate ends of nascent nuclear pre-mRNAs shortly after initiation. It is catalyzed by guanylyl- and methyltransferases, which are nuclear enzymes, but most animal viruses that replicate in the cytoplasm contain similar activities and produce capped mRNAs. Caps are retained during processing of nuclear transcripts and serve as stabilizing elements on both premRNAs in the nucleus (Green et al., Cell 32, 681-694, 1983) and mRNAs in the cytoplasm (Furuichi et al., Nature 266, 235-239, 1977). The presence of the cap markedly enhances translation by promoting initiation complex formation. In addition, structural analogs of the cap, e.g. m7G(5’)p, inhibit the attachment of 40s ribosomal subunits to capped mRNAs. Thus recognition of the capped end of mRNA by specific protein(s) is important for gene expression. However, cap involvement in mRNA function is not inevitable because the mRNAs of poliovirus and several other eukaryotic viruses lack caps (see Banerjee, Microbial. Reviews 44, 175-205, 1980). identification and Biochemical Properties of Cap Binding Proteins (CBP) Proteins that interact with the 5’ end of mRNA can be demonstrated in a variety of cell-free protein synthesizing systems. Cap binding proteins in mammalian systems have been identified by a chemical crosslinking procedure that covalently attaches cap-vicinal proteins to oxidized mRNA via the 5’-m7G (Sonenberg et al., PNAS 75, 4843-4847, 1978). By this method, a single cap specific polypeptide of about 24,000 dalton is detected in crude total factor preparations and in partially purified preparations of elF-3 and elF-46. This cap binding polypeptide (CBP I) is also the major radiolabeled polypeptide obtained after photoreaction of reticulocyte initiation factors with the cap analog, [Y-~*P]- [4-(benzophenyl) methylamidol-7-methylguanosine-5’-triphosphate (BP-m7GTP) (Pazelt et al., Nucl. Acids Res. 77, 5821-5835, 1982). Highly purified CBP I stimulates translation of capped mRNAs but not uncapped viral mRNAs (Sonenberg et al., PNAS 76, 4345-4349, 1979). CBP I forms a salt-stable complex (CBP II) with two other polypeptides: a 200,000 dalton component of unknown function, and a 46,000 dalton polypeptide that probably corresponds to elF4-A, an initiation factor required separately for attachment of mRNA to 40s ribosomal subunits (Grifo et al., JBC 258, 5804-5810, 1983). CBP II (also called elF-4F) binds mRNA and is required for maximal translation of globin mRNA in vitro; its involvement appears to be at the level of 405 initiation complex
Minireview
formation. The identification of CBP II as a functional entity rather than an adventitious association of proteins is supported by its ability to restore messenger activity to capped mRNAs in extracts of poliovirus-infected HeLa cells, a function that is lost from highly purified CBP I (Trachsel et al., PNAS 77, 770-774, 1980). The translational switch from capped host mRNAs to uncapped viral templates in poliovirus-infected cells is accompanied (possibly mediated) by proteolysis of the 200,000 dalton component of CBP II, with concomitant loss of cap binding activity (Etchison et al., JBC 257, 14806-14810, 1982). The precise relationship between this proteolysis and host shut-off remains unclear, however, because the kinetics and extent of degradation do not parallel the translational loss. A better understanding of the situation may emerge from studies using poliovirus mutants that are defective in host shut-off. Besides forming functional complexes with CBP I, certain cellular proteins also interact with the cap in a reaction that is inhibited by cap analogs. This is a property of both elF-4A and elF-4B (another factor involved in mRNA joining to 40s subunits), and is detectable by crosslinking, but only in the presence of ATP and CBP I (Grifo et al., op. cit.; Edery et al., JBC 258, 11398-11403, 1983). These results imply that CBP I binding to the cap is a primary event that facilitates ATP-dependent interactions by other, possibly related, polypeptides positioned near the 5’end of mRNA. This is an interesting possibility, given that elF-4A and CBP II have each been found to catalyze an RNAdependent hydrolysis of ATP, a reaction that is capindependent and stimulated by elF-4B (Grifo et al., JBC 259, 8648-8654, 1984). Possible Roles of CBP in Translational Initiation The involvement of CBP in translational initiation is not well-understood. Based on the data currently available, however, a general working model can be formulated. According to this model (shown in the figure), the first step in the initiation of translation is the direct binding of CBP I (or CBP II) to the 5’-end of mRNA. Initiation factors elF-4A and elF-48 then associate with CBP and, by using the energy generated by ATP hydrolysis, facilitate attachment to mRNA of a preinitiation complex consisting of the 40s ribosomal subunit, ternary complex (met-tRNApetGTP: elF-2), and other initiation factors. Among these factors is elF-3, a multi-subunit protein that associates with CBP at low ionic strength-an interaction thought to stabilize 40s initiation complexes in preparation for elF-5 mediated joining of 60s ribosomal subunits. The 80s ribosome is usually assembled after the smaller complex has moved from the 5’ end of the mRNA to the initiator AUG. Like 5’terminal attachment, this repositioning requires ATP hydrolysis (Kozak, Microbial. Reviews 47, l-45, 1983) and may include an “unwinding” of the 5’-leader sequence. Are CBP involved in this unwinding? There is no direct evidence that ATP-dependent RNA unwinding activity is associated with CBP I or CBP II, either individually or in the presence of initiation factors. Nevertheless, certain
Cell 224
5’ m7GA
’ BUG
3’
*Primary recognltlon of mRNA 5’ end I I I
3’
AUG
Attachment of elFs (W and 40s ribosamal ternary complex 3’
II
“Unwindina of mRNA 5’ leader-(?)
Migrahon initiation
*Putative
functions
of complex
of CBP
findings with mFiNAs having relaxed secondary structures [either alfalfa mosaic virus (AMV) RNA 4, which has a naturally occurring AT-rich Y-untranslated leader sequence, or reovirus mRNA, whose base pairing ability has been reduced artificially by the incorporation of inosine residues] are consistent with a cap-dependent, CBPmediated unwinding step in the above initiation scheme. For example, AMV RNA 4 can be translated in extracts of poliovirus-infected HeLa cells in which CBP have been inactivated (Sonenberg et al., MC6 2, 1633-1638, 1982). Furthermore, the in vitro messenger activity of this RNA and of inosine-substituted reovirus mRNA is relatively resistant to elevated levels of K+ that inhibit translation of other capped mRNAs. Addition of CBP II can restore messenger activity to these other RNAs, possibly by unwinding salt-stabilized base pairs in the cap-proximal region (Edery et al., Biochemistry 23, 2456-2462, 1984). Inosine-substituted mRNA appears to require ATP for formation of stable 40s initiation complexes. While preparations of crude reticulocyte initiation factors allow ATPindependent cross-linking of inosine-substituted (but not native) reovirus mRNA to presumptive elF-4A and 48 polypeptides (Lee et al., JBC 258,707-710, 1983), similar analyses done with highly purified factors demonstrate a requirement for ATP hydrolysis (Tahara et al., JBC 258, 11350-11353, 1983). From these data, it can be inferred that the putative CBP-mediated, ATP-dependent denaturation of mRNA during initiation must occur after 5’terminal attachment of the 40s initiation complex, possibly during complex repositioning at the initiator codon.
Evidence for Nontranslational Roles of CBP Localization of CBP in the nucleus has been documented recently; earlier attempts to detect them by chemical crosslinking proved unsuccessful because there are reaction inhibitors in subcellular nuclear fractions. A different approach, based on photoaffinity labeling with [Y-~~P]-BPm7GTP revealed three major polypeptides of 120,000, 89,000, and 80,000 dalton in HeLa cell nuclei (Patzelt et al., op. cit.). These CBP were resistant to extraction with detergents and nucleases, a property consistent with a nuclear location. Association of CBP with the nucleus is perhaps not surprising in light of recent evidence implicating the cap structure in molecular events thought to occur in the nuclear matrix. One of these events is pre-mRNA processing. Although m7GpppN termini are unique to mRNAs, a similar trimethylated 5’-structure, m:,2,7GpppN is present on another class of RNA polymerase II products, the U series of small nuclear RNAs (Reddy et al., JBC 249, 6486-6494,1974). One member of the series, Ul snRNA, appears to function in the alignment of mRNA 5’ splice sites, a process that requires an intact trimethylated 5’end (Kramer et al., Cell 38, 299-307, 1984). Additional evidence for involvement of the cap in pre-mRNA processing comes from the work of Konarska et al. (Cell 38, 731-736, 1984), who have found that model mRNA substrates are more efficiently spliced in a HeLa cell extract if they contain a capped 5’-end, and that the splicing reaction is inhibited by cap analogs. A positive correlation between the presence of cap and the efficiency of processing of histone mRNA 3’-ends has also been reported recently (Georgiev et al., Nucl. Acids Res. 72, 8539-8551, 1984). There is some evidence linking caps and CBP to transcriptional events. The same photoaffinity probe used to identify the nuclear CBP was found to label the PB2 polypeptide in influenzavirions (Blaas et al., Nucl. Acids Res. 70, 4803-4812, 1982). This protein functions in the initiation of viral mRNA synthesis by cleaving short, 5’capped primers from preformed heterologous transcripts (Ulmanen et al., J. Virol. 45,27-35,1983). Capped primer dependent initiation of transcription could have a cellular counterpart in trypanosome mRNA synthesis, which has been shown to be discontinuous, i.e., a capped 5’ leader common to many mRNAs is transcribed separately from regions not linked to the structural genes (Milhausen et al., Cell 38, 721-729, 1984). These examples show that cap recognition by CBP may be essential for the formation of mRNA-containing nucleoprotein complexes that function in nontranslational contexts. Continued exploration of the potential roles of caps and cap binding proteins in transcriptional initiation, premRNA processing and mRNA nucleocytoplasmic transport may provide new insights into the multilevel control of eukaryotic gene expression.
February
1985, Copyright
0 1985 by MIT
mRNA Cap Binding Proteins: Essential Factors for Initiating Translation Aaron Roche Roche Nutley,
J. Shatkin Institute of Molecular Research Center New Jersey 07110
Biology
The presence of a 5’-terminal “cap:’ m7G(5’)ppp(5’)N, is a nearly ubiquitous feature of eukaryotic mRNAs. Capping is an early reaction, occurring at the 5’-triphosphate ends of nascent nuclear pre-mRNAs shortly after initiation. It is catalyzed by guanylyl- and methyltransferases, which are nuclear enzymes, but most animal viruses that replicate in the cytoplasm contain similar activities and produce capped mRNAs. Caps are retained during processing of nuclear transcripts and serve as stabilizing elements on both premRNAs in the nucleus (Green et al., Cell 32, 681-694, 1983) and mRNAs in the cytoplasm (Furuichi et al., Nature 266, 235-239, 1977). The presence of the cap markedly enhances translation by promoting initiation complex formation. In addition, structural analogs of the cap, e.g. m7G(5’)p, inhibit the attachment of 40s ribosomal subunits to capped mRNAs. Thus recognition of the capped end of mRNA by specific protein(s) is important for gene expression. However, cap involvement in mRNA function is not inevitable because the mRNAs of poliovirus and several other eukaryotic viruses lack caps (see Banerjee, Microbial. Reviews 44, 175-205, 1980). identification and Biochemical Properties of Cap Binding Proteins (CBP) Proteins that interact with the 5’ end of mRNA can be demonstrated in a variety of cell-free protein synthesizing systems. Cap binding proteins in mammalian systems have been identified by a chemical crosslinking procedure that covalently attaches cap-vicinal proteins to oxidized mRNA via the 5’-m7G (Sonenberg et al., PNAS 75, 4843-4847, 1978). By this method, a single cap specific polypeptide of about 24,000 dalton is detected in crude total factor preparations and in partially purified preparations of elF-3 and elF-46. This cap binding polypeptide (CBP I) is also the major radiolabeled polypeptide obtained after photoreaction of reticulocyte initiation factors with the cap analog, [Y-~*P]- [4-(benzophenyl) methylamidol-7-methylguanosine-5’-triphosphate (BP-m7GTP) (Pazelt et al., Nucl. Acids Res. 77, 5821-5835, 1982). Highly purified CBP I stimulates translation of capped mRNAs but not uncapped viral mRNAs (Sonenberg et al., PNAS 76, 4345-4349, 1979). CBP I forms a salt-stable complex (CBP II) with two other polypeptides: a 200,000 dalton component of unknown function, and a 46,000 dalton polypeptide that probably corresponds to elF4-A, an initiation factor required separately for attachment of mRNA to 40s ribosomal subunits (Grifo et al., JBC 258, 5804-5810, 1983). CBP II (also called elF-4F) binds mRNA and is required for maximal translation of globin mRNA in vitro; its involvement appears to be at the level of 405 initiation complex
Minireview
formation. The identification of CBP II as a functional entity rather than an adventitious association of proteins is supported by its ability to restore messenger activity to capped mRNAs in extracts of poliovirus-infected HeLa cells, a function that is lost from highly purified CBP I (Trachsel et al., PNAS 77, 770-774, 1980). The translational switch from capped host mRNAs to uncapped viral templates in poliovirus-infected cells is accompanied (possibly mediated) by proteolysis of the 200,000 dalton component of CBP II, with concomitant loss of cap binding activity (Etchison et al., JBC 257, 14806-14810, 1982). The precise relationship between this proteolysis and host shut-off remains unclear, however, because the kinetics and extent of degradation do not parallel the translational loss. A better understanding of the situation may emerge from studies using poliovirus mutants that are defective in host shut-off. Besides forming functional complexes with CBP I, certain cellular proteins also interact with the cap in a reaction that is inhibited by cap analogs. This is a property of both elF-4A and elF-4B (another factor involved in mRNA joining to 40s subunits), and is detectable by crosslinking, but only in the presence of ATP and CBP I (Grifo et al., op. cit.; Edery et al., JBC 258, 11398-11403, 1983). These results imply that CBP I binding to the cap is a primary event that facilitates ATP-dependent interactions by other, possibly related, polypeptides positioned near the 5’end of mRNA. This is an interesting possibility, given that elF-4A and CBP II have each been found to catalyze an RNAdependent hydrolysis of ATP, a reaction that is capindependent and stimulated by elF-4B (Grifo et al., JBC 259, 8648-8654, 1984). Possible Roles of CBP in Translational Initiation The involvement of CBP in translational initiation is not well-understood. Based on the data currently available, however, a general working model can be formulated. According to this model (shown in the figure), the first step in the initiation of translation is the direct binding of CBP I (or CBP II) to the 5’-end of mRNA. Initiation factors elF-4A and elF-48 then associate with CBP and, by using the energy generated by ATP hydrolysis, facilitate attachment to mRNA of a preinitiation complex consisting of the 40s ribosomal subunit, ternary complex (met-tRNApetGTP: elF-2), and other initiation factors. Among these factors is elF-3, a multi-subunit protein that associates with CBP at low ionic strength-an interaction thought to stabilize 40s initiation complexes in preparation for elF-5 mediated joining of 60s ribosomal subunits. The 80s ribosome is usually assembled after the smaller complex has moved from the 5’ end of the mRNA to the initiator AUG. Like 5’terminal attachment, this repositioning requires ATP hydrolysis (Kozak, Microbial. Reviews 47, l-45, 1983) and may include an “unwinding” of the 5’-leader sequence. Are CBP involved in this unwinding? There is no direct evidence that ATP-dependent RNA unwinding activity is associated with CBP I or CBP II, either individually or in the presence of initiation factors. Nevertheless, certain
Cell 224
5’ m7GA
’ BUG
3’
*Primary recognltlon of mRNA 5’ end I I I
3’
AUG
Attachment of elFs (W and 40s ribosamal ternary complex 3’
II
“Unwindina of mRNA 5’ leader-(?)
Migrahon initiation
*Putative
functions
of complex
of CBP
findings with mFiNAs having relaxed secondary structures [either alfalfa mosaic virus (AMV) RNA 4, which has a naturally occurring AT-rich Y-untranslated leader sequence, or reovirus mRNA, whose base pairing ability has been reduced artificially by the incorporation of inosine residues] are consistent with a cap-dependent, CBPmediated unwinding step in the above initiation scheme. For example, AMV RNA 4 can be translated in extracts of poliovirus-infected HeLa cells in which CBP have been inactivated (Sonenberg et al., MC6 2, 1633-1638, 1982). Furthermore, the in vitro messenger activity of this RNA and of inosine-substituted reovirus mRNA is relatively resistant to elevated levels of K+ that inhibit translation of other capped mRNAs. Addition of CBP II can restore messenger activity to these other RNAs, possibly by unwinding salt-stabilized base pairs in the cap-proximal region (Edery et al., Biochemistry 23, 2456-2462, 1984). Inosine-substituted mRNA appears to require ATP for formation of stable 40s initiation complexes. While preparations of crude reticulocyte initiation factors allow ATPindependent cross-linking of inosine-substituted (but not native) reovirus mRNA to presumptive elF-4A and 48 polypeptides (Lee et al., JBC 258,707-710, 1983), similar analyses done with highly purified factors demonstrate a requirement for ATP hydrolysis (Tahara et al., JBC 258, 11350-11353, 1983). From these data, it can be inferred that the putative CBP-mediated, ATP-dependent denaturation of mRNA during initiation must occur after 5’terminal attachment of the 40s initiation complex, possibly during complex repositioning at the initiator codon.
Evidence for Nontranslational Roles of CBP Localization of CBP in the nucleus has been documented recently; earlier attempts to detect them by chemical crosslinking proved unsuccessful because there are reaction inhibitors in subcellular nuclear fractions. A different approach, based on photoaffinity labeling with [Y-~~P]-BPm7GTP revealed three major polypeptides of 120,000, 89,000, and 80,000 dalton in HeLa cell nuclei (Patzelt et al., op. cit.). These CBP were resistant to extraction with detergents and nucleases, a property consistent with a nuclear location. Association of CBP with the nucleus is perhaps not surprising in light of recent evidence implicating the cap structure in molecular events thought to occur in the nuclear matrix. One of these events is pre-mRNA processing. Although m7GpppN termini are unique to mRNAs, a similar trimethylated 5’-structure, m:,2,7GpppN is present on another class of RNA polymerase II products, the U series of small nuclear RNAs (Reddy et al., JBC 249, 6486-6494,1974). One member of the series, Ul snRNA, appears to function in the alignment of mRNA 5’ splice sites, a process that requires an intact trimethylated 5’end (Kramer et al., Cell 38, 299-307, 1984). Additional evidence for involvement of the cap in pre-mRNA processing comes from the work of Konarska et al. (Cell 38, 731-736, 1984), who have found that model mRNA substrates are more efficiently spliced in a HeLa cell extract if they contain a capped 5’-end, and that the splicing reaction is inhibited by cap analogs. A positive correlation between the presence of cap and the efficiency of processing of histone mRNA 3’-ends has also been reported recently (Georgiev et al., Nucl. Acids Res. 72, 8539-8551, 1984). There is some evidence linking caps and CBP to transcriptional events. The same photoaffinity probe used to identify the nuclear CBP was found to label the PB2 polypeptide in influenzavirions (Blaas et al., Nucl. Acids Res. 70, 4803-4812, 1982). This protein functions in the initiation of viral mRNA synthesis by cleaving short, 5’capped primers from preformed heterologous transcripts (Ulmanen et al., J. Virol. 45,27-35,1983). Capped primer dependent initiation of transcription could have a cellular counterpart in trypanosome mRNA synthesis, which has been shown to be discontinuous, i.e., a capped 5’ leader common to many mRNAs is transcribed separately from regions not linked to the structural genes (Milhausen et al., Cell 38, 721-729, 1984). These examples show that cap recognition by CBP may be essential for the formation of mRNA-containing nucleoprotein complexes that function in nontranslational contexts. Continued exploration of the potential roles of caps and cap binding proteins in transcriptional initiation, premRNA processing and mRNA nucleocytoplasmic transport may provide new insights into the multilevel control of eukaryotic gene expression.