1607253
Enzyme 1990;44:310-319
Translational Regulation of the Immunoglobulin Heavy-Chain Binding Protein mRNA Dennis G. Macejak, Peter Sarnow Department of Biochemistry, Biophysics and Genetics, Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver, Colo., USA
Key Words. Internal initiation • Immunoglobulin heavy-chain binding protein • Glucose-regulated protein 78 • Poliovirus • Luciferase ■ RNA transfection. Abstract. Translation of the mRNA encoding the immunoglobulin heavy-chain binding protein (BiP) is enhanced in poliovirus-infected cells at a time when translation of host cell mRNAs is inhibited. To test whether the mRNA of BiP is translated by internal ribosome binding, like picomaviral RNAs, we constructed plasmids for the expression of dicistronic hybrid RNAs containing the 5' noncoding region (5'NCR) of BiP as an intercistronic spacer element between two cistrons. Expression of these dicistronic mRNAs in mammalian cells resulted in efficient translation of both cistrons, demonstrating that the 5'NCR of BiP can confer internal ribosome binding to a heterologous RNA. This result suggests that the mRNA encoding BiP is bifunctional and can be translated by an internal ribosome-binding mecha nism, in addition to the conventional cap-dependent scanning mechanism. This is the first demonstration of a cellular mRNA that can be translated by internal ribosome binding, and implies that this may be a mechanism for cellular translational regulation.
The immunoglobulin heavy-chain bind ing protein (BiP) is a soluble 78-kD protein residing in the lumen of the endoplasmic reticulum (ER) [1,2], and it is retained there by a specific receptor-mediated retention system [3, 4]. BiP was first identified in Bcell precursors as a protein transiently asso ciated with the immunoglobulin heavy chain before assembly with the light chain [2], Cloning and sequencing of the gene encoding
BiP has revealed that BiP is identical to the glucose-regulated protein (GRP) 78 [5], Fur thermore, BiP shares 60% amino acid ho mology with heat-shock protein 70 [5], Proposed functions for BiP include facili tating the proper folding and assembly of nascent proteins in the ER before their sub sequent transport to the Golgi apparatus [2, 6] and the scavenging of misfolded proteins in the ER [7], For example, mutant or malfolded secretory proteins have been ob served to be more tightly associated with BiP Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 11:38:18 AM
Introduction
than their normal, properly folded homo logues; perhaps this association prevents their export out of the ER. Although BiP is an abundant protein in the ER of virtually all eukaryotic cells inves tigated to date, its synthesis is further in duced in response to a variety of stresses such as glucose starvation [8], tunicamycin or glucosamine treatment [9, 10], paramyxo virus infection [11], and treatment with cal cium ionophores or calcium-chelating agents [12], Under each of these conditions, in creased transcriptional activity of the BiP gene and increased steady-state levels of BiP mRNA are observed. Moreover, the expres sion of BiP and GRP94 (another GRP) genes is always coordinately regulated under these transcriptional induction conditions [13]. We have observed that the synthesis of BiP can be regulated at the posttranscriptional level as well. Specifically, the mRNA encoding BiP is translated at an enhanced level in poliovirus-infected cells at a time when cap-dependent translation of host cell mRNA is inhibited [14], We have found that the 5' noncoding region (5'NCR) of BiP mRNA can initiate translation by an internal ribosome-binding mechanism. The observa tions leading to the discovery of this novel translational regulation of BiP mRNA will be summarized here, and their implications for the regulation of expression of the BiP gene will be discussed.
Results
Translational Regulation of BiP mRNA in Poliovirus-Infected Cells Initially, we studied early events in poliovirus-infected cells with the help of two de fined poliovirus mutants, 3NC202 and
311
2B201. Mutant 3NC202 bears an 8-nucleotide (nt) insertion in the 3'NCR and is tem perature-sensitive for RNA synthesis [15]. Mutant 2B201 contains a 6-nt insertion in the region encoding polypeptide 2B and also fails to synthesize normal amounts of viral RNA [16]. Because cells infected with either mu tant synthesize only low levels of viral RNA, they were used to study the function of viral RNA molecules and their translation prod ucts at times early in the infectious cycle. Specifically, we were interested in the proteolytic processing of the viral polypro tein at times early in infection. Therefore, human HeLa suspension cells were infected with wild-type poliovirus, mutant 2B201 vi rus at 37 °C, or mutant 3NC202 virus at 39.5 °C (the nonpermissive condition for vi ral replication of this mutant). Three hours after infection, the cells were labeled with 35S-methionine for 30 min, soluble extracts were prepared, and labeled proteins were analyzed by SDS-PAGE. Figure 1 shows that extracts obtained from wild-type-infected cells displayed a typical pattern of viral pro tein precursors and end products (lane WT) and an efficient inhibition of host cell trans lation (compare WT and the MOCK lanes). This inhibition of host cell translation in po liovirus-infected cells is due to a proteolytic inactivation of the cap-binding protein com plex eIF-4F [17], Extracts obtained from mutant-infected cells (lanes 3NC202 and 2B201) showed an inhibition of host-cell translation, even in the absence of signifi cant viral replication. In addition to viral protein precursors, an 80-kD protein (de noted by the arrow) could be detected in extracts derived from mutant-infected cells, Interestingly, this protein could also be seen in uninfected cell extracts, and in wild-type poliovirus-infected extracts at times early afDownloaded by: University of Exeter 144.173.6.94 - 1/26/2020 11:38:18 AM
Translational Regulation of BiP mRNA
Macejak/Samow
CM
O
i_
O O
CM °
5
CO
CM
t
13CD-
2
CÛ
r I
O O
Q. CD
4»
2C-
I
VP3-
Fig. 1. Detection of a cellular 80-kD protein in cells infected with different poliovirus mutants. HeLa cells grown in suspension culture were infected with wild-type (WT) and mutant (3NC202, 2B201) polio viruses and labeled with 35S-methionine. Soluble ex tracts were prepared and analyzed by SDS-PAGE. In addition, labeled extracts from 3NC202-infected cells were immunoprecipitated with a monoclonal anti body directed against BiP [2] and analyzed by SDSPAGE (BiP). An autoradiograph of the gel is shown. The arrow denotes the 80-kD protein.
ter infection [14]. Comparison by limited proteolysis of the 80-kD proteins obtained from mutant- and mock-infected cells re vealed a strong similarity or identity of the 80-kD proteins, suggesting that the 80-kD protein was translated from a cellular mRNA at a time when overall cap-depen dent host cell translation was inhibited.
Curiously, we noted that the synthesis of the 80-kD protein could not be detected in HeLa cells grown as monolayer cultures, un less they were infected with poliovirus [14]. Comparison of the ingredients in the differ ent media used to grow suspension cells and monolayer culture revealed that in suspen sion, cells were grown in medium containing one fifth the concentration of glucose. At that time, we studied a report describing the biochemical properties of GRP78 [18]. We noted that the limited proteolysis map of GRP78, a protein induced during glucose starvation of mammalian cells, looked very similar to that of the 80-kD protein. To test whether the cellular 80-kD protein was iden tical to GRP78, HeLa cells grown as monolayer cultures were deprived of glucose, metabolically labeled with 35S-methionine and extracts were analyzed by one- and twodimensional gel electrophoresis [14]. The re sults showed that glucose deprivation in duced the synthesis of a protein that dis played the same 80,000 molecular weight and the same isoelectric point as the cellular 80-kD protein seen early in poliovirus-in fected cells [14], indicating that the cellular 80-kD protein was identical to GRP78. GRP78 is identical to the immunoglobu lin heavy-chain BiP [5] and, therefore, we used a monoclonal antibody directed against BiP [2] to immunoprecipitate extracts ob tained from 3NC202-infected cells. Figure 1 (lane BiP) shows that an 80-kD protein could be immunoprecipitated from this extract, verifying its identity with GRP78/BÌP. The next question was whether the in creased level of the BiP protein was due to increased transcription of the cellular BiP gene during poliovirus infection. Northern analysis showed that the steady-state level of BiP mRNA was the same in uninfected and Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 11:38:18 AM
312
Translational Regulation of BiP mRNA
313
infected cells. Thus, the increased amount of BiP observed by pulse-labeling in infected cells (fig. 1 ) was probably due to an increased rate of translation of BiP mRNA rather than an increased level of mRNA. This was unex pected because the regulation of BiP mRNA was reported to occur at the transcriptional level during various stress situations of the cell such as glucose starvation [13], infection by paramyxovirus [11], and transformation by Rous sarcoma virus [19]. In contrast, we found that BiP was induced at the transla tional level in poliovirus-infected cells at a time when cap-dependent translation of cel lular mRNAs was inhibited.
Fig. 2. Enhanced translation of hybrid RNA mole cules containing the 5'NCR of BiP, after transfection into poliovirus-infected HeLa cells. 1 pg of capped RNAs containing the 5'NCR of LUC or BiP (BiPLUC) linked to the LUC-coding region were directly transfected into HeLa cells [21] that had been in fected with wild-type poliovirus. Soluble extracts were prepared at different times after infection and the amount of LUC activity was determined.
slight accumulation of translation product from capped RNAs bearing the 5'NCR of LUC from 1 to 3 h after infection, which declined slightly at 5 h after infection due to the rapid turnover of the unstable LUC. However, RNAs bearing the 5'NCR of BiP (BiP-LUC) displayed a more dramatic in crease in translation from 1 to 3 h after infec tion, mimicking the effects seen in infected cells (fig. 1). These findings indicated that the en hanced translation of BiP mRNA in poliovi rus-infected cells, a condition in which elF4F is nonfunctional, was conferred by the 5'NCR of the BiP gene. Furthermore, this Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 11:38:18 AM
5'NCR of the BiP mRNA Is Sufficient to Confer Enhanced Translation to a Heterologous mRNA in Poliovirus-Infected Cells Our next aim was to test whether the 5'NCR of BiP mRNA was sufficient for the enhanced translation of the BiP mRNA in poliovirus-infected cells. We tested this by direct transfection of hybrid RNA molecules into mammalian cells. Plasmids were constructed from which RNA molecules could be synthesized in vitro by T7 RNA polymerase. These RNAs con tained m7GpppN-capped 5'NCRs of luciferase (LUC) and BiP, respectively, upstream of the coding region for the firefly LUC gene [20], The amounts of the two RNA species were quantitated by Northern analysis, and these RNAs were transfected directly into mammalian cells [21; Hambidge and Sarnow, unpublished data]. The translation of the two RNA species was indistinguishable when transfected into uninfected HeLa cells (data not shown). However, the translation of the two RNA species was distinguishable in poliovirus-infected cells. Figure 2 shows a
Macejak/Samow
314
enhanced translation could be tested by di rect transfection of RNAs into the cells, demonstrating that this effect was a posttranscriptional event, probably translational regulation.
3 60,000
C/BiP/L
onî
Enzyme 1990;44:310-319
Translational Regulation of the Immunoglobulin Heavy-Chain Binding Protein mRNA Dennis G. Macejak, Peter Sarnow Department of Biochemistry, Biophysics and Genetics, Department of Microbiology and Immunology, University of Colorado Health Sciences Center, Denver, Colo., USA
Key Words. Internal initiation • Immunoglobulin heavy-chain binding protein • Glucose-regulated protein 78 • Poliovirus • Luciferase ■ RNA transfection. Abstract. Translation of the mRNA encoding the immunoglobulin heavy-chain binding protein (BiP) is enhanced in poliovirus-infected cells at a time when translation of host cell mRNAs is inhibited. To test whether the mRNA of BiP is translated by internal ribosome binding, like picomaviral RNAs, we constructed plasmids for the expression of dicistronic hybrid RNAs containing the 5' noncoding region (5'NCR) of BiP as an intercistronic spacer element between two cistrons. Expression of these dicistronic mRNAs in mammalian cells resulted in efficient translation of both cistrons, demonstrating that the 5'NCR of BiP can confer internal ribosome binding to a heterologous RNA. This result suggests that the mRNA encoding BiP is bifunctional and can be translated by an internal ribosome-binding mecha nism, in addition to the conventional cap-dependent scanning mechanism. This is the first demonstration of a cellular mRNA that can be translated by internal ribosome binding, and implies that this may be a mechanism for cellular translational regulation.
The immunoglobulin heavy-chain bind ing protein (BiP) is a soluble 78-kD protein residing in the lumen of the endoplasmic reticulum (ER) [1,2], and it is retained there by a specific receptor-mediated retention system [3, 4]. BiP was first identified in Bcell precursors as a protein transiently asso ciated with the immunoglobulin heavy chain before assembly with the light chain [2], Cloning and sequencing of the gene encoding
BiP has revealed that BiP is identical to the glucose-regulated protein (GRP) 78 [5], Fur thermore, BiP shares 60% amino acid ho mology with heat-shock protein 70 [5], Proposed functions for BiP include facili tating the proper folding and assembly of nascent proteins in the ER before their sub sequent transport to the Golgi apparatus [2, 6] and the scavenging of misfolded proteins in the ER [7], For example, mutant or malfolded secretory proteins have been ob served to be more tightly associated with BiP Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 11:38:18 AM
Introduction
than their normal, properly folded homo logues; perhaps this association prevents their export out of the ER. Although BiP is an abundant protein in the ER of virtually all eukaryotic cells inves tigated to date, its synthesis is further in duced in response to a variety of stresses such as glucose starvation [8], tunicamycin or glucosamine treatment [9, 10], paramyxo virus infection [11], and treatment with cal cium ionophores or calcium-chelating agents [12], Under each of these conditions, in creased transcriptional activity of the BiP gene and increased steady-state levels of BiP mRNA are observed. Moreover, the expres sion of BiP and GRP94 (another GRP) genes is always coordinately regulated under these transcriptional induction conditions [13]. We have observed that the synthesis of BiP can be regulated at the posttranscriptional level as well. Specifically, the mRNA encoding BiP is translated at an enhanced level in poliovirus-infected cells at a time when cap-dependent translation of host cell mRNA is inhibited [14], We have found that the 5' noncoding region (5'NCR) of BiP mRNA can initiate translation by an internal ribosome-binding mechanism. The observa tions leading to the discovery of this novel translational regulation of BiP mRNA will be summarized here, and their implications for the regulation of expression of the BiP gene will be discussed.
Results
Translational Regulation of BiP mRNA in Poliovirus-Infected Cells Initially, we studied early events in poliovirus-infected cells with the help of two de fined poliovirus mutants, 3NC202 and
311
2B201. Mutant 3NC202 bears an 8-nucleotide (nt) insertion in the 3'NCR and is tem perature-sensitive for RNA synthesis [15]. Mutant 2B201 contains a 6-nt insertion in the region encoding polypeptide 2B and also fails to synthesize normal amounts of viral RNA [16]. Because cells infected with either mu tant synthesize only low levels of viral RNA, they were used to study the function of viral RNA molecules and their translation prod ucts at times early in the infectious cycle. Specifically, we were interested in the proteolytic processing of the viral polypro tein at times early in infection. Therefore, human HeLa suspension cells were infected with wild-type poliovirus, mutant 2B201 vi rus at 37 °C, or mutant 3NC202 virus at 39.5 °C (the nonpermissive condition for vi ral replication of this mutant). Three hours after infection, the cells were labeled with 35S-methionine for 30 min, soluble extracts were prepared, and labeled proteins were analyzed by SDS-PAGE. Figure 1 shows that extracts obtained from wild-type-infected cells displayed a typical pattern of viral pro tein precursors and end products (lane WT) and an efficient inhibition of host cell trans lation (compare WT and the MOCK lanes). This inhibition of host cell translation in po liovirus-infected cells is due to a proteolytic inactivation of the cap-binding protein com plex eIF-4F [17], Extracts obtained from mutant-infected cells (lanes 3NC202 and 2B201) showed an inhibition of host-cell translation, even in the absence of signifi cant viral replication. In addition to viral protein precursors, an 80-kD protein (de noted by the arrow) could be detected in extracts derived from mutant-infected cells, Interestingly, this protein could also be seen in uninfected cell extracts, and in wild-type poliovirus-infected extracts at times early afDownloaded by: University of Exeter 144.173.6.94 - 1/26/2020 11:38:18 AM
Translational Regulation of BiP mRNA
Macejak/Samow
CM
O
i_
O O
CM °
5
CO
CM
t
13CD-
2
CÛ
r I
O O
Q. CD
4»
2C-
I
VP3-
Fig. 1. Detection of a cellular 80-kD protein in cells infected with different poliovirus mutants. HeLa cells grown in suspension culture were infected with wild-type (WT) and mutant (3NC202, 2B201) polio viruses and labeled with 35S-methionine. Soluble ex tracts were prepared and analyzed by SDS-PAGE. In addition, labeled extracts from 3NC202-infected cells were immunoprecipitated with a monoclonal anti body directed against BiP [2] and analyzed by SDSPAGE (BiP). An autoradiograph of the gel is shown. The arrow denotes the 80-kD protein.
ter infection [14]. Comparison by limited proteolysis of the 80-kD proteins obtained from mutant- and mock-infected cells re vealed a strong similarity or identity of the 80-kD proteins, suggesting that the 80-kD protein was translated from a cellular mRNA at a time when overall cap-depen dent host cell translation was inhibited.
Curiously, we noted that the synthesis of the 80-kD protein could not be detected in HeLa cells grown as monolayer cultures, un less they were infected with poliovirus [14]. Comparison of the ingredients in the differ ent media used to grow suspension cells and monolayer culture revealed that in suspen sion, cells were grown in medium containing one fifth the concentration of glucose. At that time, we studied a report describing the biochemical properties of GRP78 [18]. We noted that the limited proteolysis map of GRP78, a protein induced during glucose starvation of mammalian cells, looked very similar to that of the 80-kD protein. To test whether the cellular 80-kD protein was iden tical to GRP78, HeLa cells grown as monolayer cultures were deprived of glucose, metabolically labeled with 35S-methionine and extracts were analyzed by one- and twodimensional gel electrophoresis [14]. The re sults showed that glucose deprivation in duced the synthesis of a protein that dis played the same 80,000 molecular weight and the same isoelectric point as the cellular 80-kD protein seen early in poliovirus-in fected cells [14], indicating that the cellular 80-kD protein was identical to GRP78. GRP78 is identical to the immunoglobu lin heavy-chain BiP [5] and, therefore, we used a monoclonal antibody directed against BiP [2] to immunoprecipitate extracts ob tained from 3NC202-infected cells. Figure 1 (lane BiP) shows that an 80-kD protein could be immunoprecipitated from this extract, verifying its identity with GRP78/BÌP. The next question was whether the in creased level of the BiP protein was due to increased transcription of the cellular BiP gene during poliovirus infection. Northern analysis showed that the steady-state level of BiP mRNA was the same in uninfected and Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 11:38:18 AM
312
Translational Regulation of BiP mRNA
313
infected cells. Thus, the increased amount of BiP observed by pulse-labeling in infected cells (fig. 1 ) was probably due to an increased rate of translation of BiP mRNA rather than an increased level of mRNA. This was unex pected because the regulation of BiP mRNA was reported to occur at the transcriptional level during various stress situations of the cell such as glucose starvation [13], infection by paramyxovirus [11], and transformation by Rous sarcoma virus [19]. In contrast, we found that BiP was induced at the transla tional level in poliovirus-infected cells at a time when cap-dependent translation of cel lular mRNAs was inhibited.
Fig. 2. Enhanced translation of hybrid RNA mole cules containing the 5'NCR of BiP, after transfection into poliovirus-infected HeLa cells. 1 pg of capped RNAs containing the 5'NCR of LUC or BiP (BiPLUC) linked to the LUC-coding region were directly transfected into HeLa cells [21] that had been in fected with wild-type poliovirus. Soluble extracts were prepared at different times after infection and the amount of LUC activity was determined.
slight accumulation of translation product from capped RNAs bearing the 5'NCR of LUC from 1 to 3 h after infection, which declined slightly at 5 h after infection due to the rapid turnover of the unstable LUC. However, RNAs bearing the 5'NCR of BiP (BiP-LUC) displayed a more dramatic in crease in translation from 1 to 3 h after infec tion, mimicking the effects seen in infected cells (fig. 1). These findings indicated that the en hanced translation of BiP mRNA in poliovi rus-infected cells, a condition in which elF4F is nonfunctional, was conferred by the 5'NCR of the BiP gene. Furthermore, this Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 11:38:18 AM
5'NCR of the BiP mRNA Is Sufficient to Confer Enhanced Translation to a Heterologous mRNA in Poliovirus-Infected Cells Our next aim was to test whether the 5'NCR of BiP mRNA was sufficient for the enhanced translation of the BiP mRNA in poliovirus-infected cells. We tested this by direct transfection of hybrid RNA molecules into mammalian cells. Plasmids were constructed from which RNA molecules could be synthesized in vitro by T7 RNA polymerase. These RNAs con tained m7GpppN-capped 5'NCRs of luciferase (LUC) and BiP, respectively, upstream of the coding region for the firefly LUC gene [20], The amounts of the two RNA species were quantitated by Northern analysis, and these RNAs were transfected directly into mammalian cells [21; Hambidge and Sarnow, unpublished data]. The translation of the two RNA species was indistinguishable when transfected into uninfected HeLa cells (data not shown). However, the translation of the two RNA species was distinguishable in poliovirus-infected cells. Figure 2 shows a
Macejak/Samow
314
enhanced translation could be tested by di rect transfection of RNAs into the cells, demonstrating that this effect was a posttranscriptional event, probably translational regulation.
3 60,000
C/BiP/L
onî
