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A site-specific mRNA-cytoplasmic protein complex in vitro
119
TIBS 13-April 1988
Journal Club A site-specific mRPIA-cytoplasmic protein complex in v/tro Ruth/vL Starzyk Site specific mRNA-protein complexes found in all known ferfitin H and L subregulate the expression of some genes at unit genes, makes up the loop and adjathe level of translation. Among the best- cent stem nucleotides (Fig. 1). Deletion studied systems to exhibit such transla- of the 28-base region eliminates iron tional regulation are prokaryotic regulation of chimeric ferritin-chlorribosomal proteins z. Some of these amphenicoi acetyltransferase or ferdtinoperons are controlled by a translational human growth hormone constructs in feedback mechanism. Here, one of the vivo3,4. Moreover, a synthetic deoxypolypepfide products selectively binds oligonucleotide with this sequence conthe mRNA to prevent further synthesis fers iron responsiveness upon a construct of some or all of the operon's proteins. otherwise unable to respond to iron4. Leibold and Munro 5 incubated cytoLimited sequence specificity and its resulting secondary structure provide the plasmic extracts from rat tissues or cultured rat hepatoma cells with a labeled target site on the mRNA molecule. In higher organisms, translational transcript of part of the 5' untranslated regulation is exemplified by iron- region of rat ferritin L-mRNA, which mediated control of ferritin, an iron- included the 28-base region. Two RNAstorage protein2. In tl:is case, a highly protein complexes, called B1 and B2, conserved 2g-base sequence in the 5' were resolved electrophoretically on untranslated region of ferritin mRNA non-denaturing polyacrylamide gels with has been implicated as a target site for the use of RNase T1 and heparin. Similar the iron-sensitive factor in vivo 3,4. results were obtained with the 5' Recently, Leibold and Munro have iden- untranslated region of rat ferritin Htified a specific cytoplasmic protein mRNA. Competition studies indicate which binds this sequence in vino s. Tiffs that the complexes are specific to the ferprotein-mRNA complex, which is ritin mRNAs. This specificity probably affected by iron, may play a role in the lies in the 28-base sequence because this iron-mediated translational regulation of sequence alone is shared by the 5' untranslated regions of rat ferritin L- and ferritin. Ferritin is found in animal, plant, fun- H-mRNAsS,7. Complex formation in response to iron gal and bacterial cells2. In vertebrates, its protein shell is composed of two types of parallels the translational activation of H subunit: heavy (H) and light (L). Upon and L mRNAs. With liver extracts from addition of iron, synthesis of both sub- rats injected with iron, B2 first units is directed by mRNAs recruited diminishes as the stored mRNA shifts to from a formerly inactive cytoplasmic the polysomes, then disappears entirely, message pool (Ref. 2, and references in and subsequently reappears as the ferriRef. 5). Genes for both subunits have tin mRNA moves back to the RNP parbeen sequenced from a number of ticles. The amount of B1, which starts eukaryotes (see references in Ref. 5). out somewhat greater than that of B2, Computer predictions suggest that the does not diminish over 16 hours. Similar first 76 nucleotides of the rather long fer- results are obtained with heart tissue. ritin 5' untranslated regions can form as With cultured rat hepatoma cells, B2 stem-loop structures. One such com- starts out higher than B 1. As with the tisputer-modeled structure is shown in Fig. sues, though, iron administration effects 1 for the rat ferritin-L mRNA 3,6. The a decrease in B2 with, interestingly, a aforementioned 28-base sequence, concomitant gain in B1. Any reciprocal relationship between B1 and B2 cannot be ascertained from the present data, R. M. Starzyk is at the Deparanent of Biology, Massachusetts Institute of Technology, Cambridge, however. In both complexes the protected RNA MA 02139, USA.
is approximately 50 bases in length. To determine the sequence, cytoplasmic extracts were chosen to produce either B1 alone (iron treatment) or both complexes, and the protected RNA was mapped with RNase T1. In. Fig. l, the protected region, which appears to be the same for both complexes, is boxed. The conserved 28-base sequence (bold letters) is completely protected and lies well within the boundaries of the 50-base area. Leibold and Munro note that at the 3' boundary the protected area might
RGU6 0 R-IJ R-U C-6 U 6 U-R 6-C U 6 C-6 U-R A
,~-- 61 (- 140)
B
U -A G-
['e-
C C C
C- G
G
C- G G A C 16 --I~ (-105)
...AA- U G -- C
A G5' - ~ - - - - - -
(-190) 1 !
GU -
C C 76 (- 125)
i~g. 1. Computer-modeled secondary s~cture of a portion of the ratferritin-L mRNA 5' untranslated region. The conserved 28-base sequence is in bold type. The region protected in the protein-mRNA complexes is boxeds. Numbers indicate positions in the sequence starting from the fu~t transcribed base6. Numbers in parentheses indicate the number of bases 5' to the AUG startcodon6. This figure is after Fig. 1 of Ref. 3 and Fig. 3 of Ref. 6. ~ ) 1088, ~lsevier Publications Cambridge 0376 - 5067/8.8/$02.00
TIBS 13-April 1988
120 extend through four additional unlabeled bases to bring the collective size of the mapped fragments up to that observed for the intact protected region. To identify binding proteins, the labeled complexes were UV crosslinked and subsequently analysed by SDS gel electrophoresis. A single complex of 100 kDa was obtained. Mowing for bound RNA, this would correspond to a protein of about 85 kDa. Proteinase K treatment prior to UV crosslinking prevented complex formation, as did excess unlabeled 5' L-mRNA. Excess unlabeled, nonspecific, competitor RNA did not affect the complex. Iron administration to rats did not change the amount of crosslinked complex formed with tissue extracts. Upon iron addition to cultured cells, however, the crosslinked complex increased in parallel with B1. This suggests that the crosslinked protein
may be a component of B1. In B2, the spatial orientation of the binding protein(s) to the RNA may not favor crosslinking. This work, then, provides the first indication in vitro that iron-sensitive cytoplasmic protein(s) may bind ferritin mRNAs at a specific site and thus regulate their translation. The most likely target is a 28-base sequence highly conserved among all known ferritin mRNAs. In the rat L-mRNA, it is located 142-169 nucleotides before the AUG start of the polypeptide subunit. This region can be drawn in a stem and loop structure as has been suggested for mRNAs of some translationallycontrolled prokaryotic genes (see Fig. 1). At present, however, there is no experimental evidence that such a structure exists. Future work on this system should elucidate the mechanism of iron-
mediated translational control of ferritin synthesis and a role, if any, for site specific mRNA-protein complexes.
References 1 Nomura, M., Gourse, R. and Baughman, G. (1984) Annu. Rev. Biochem. 53, 75-117 2 Theft, E. C. (1987)Annu. Rev. Biochem. 56, 289-315 3 Aziz, N. and Munro, H. N. (1987) Proc. Natl Acad. Sci. USA 84, 8478-8482 4 Hentze, M. W., Caughman, S. W., Rouault, T. A., Barriocanal, J. G., Dancis, A., Harford, J. B. and Klausner, R. D. (1987) Science 238, 1570-1573 5 Leibold, E. A. and Munro, H. N. (1988) Proc. Natl Acad. Sci. USA 85, 2171-2175 6 Leibold, E. A. and Munro, H. N. (1987) J. Biol. Chem. 262, 7335-7341 7 Murray, M. T., White, K. and Munro, H. N. (1987) Proc. Natl Acad. Sci. USA 84, 74387442
Features parent strains~. However, attempts to identify the suppressor gene products gave conflicting results. On the one hand, several properties of ribosomes in the strains carrying recessive suppressor new clues to an old puzzle mutations were found to be changed: • the level of ribosomal ambiguity is AncireiP. Surguchov increased4-~; • the interaction of ribosomes with antiGenetic characterization of omnipotent nonsense-suppressors sup/and sup2 in yeast biotics is altered, e.g. association conoriginally suggested that they code for termination factors, but their role in the control stant K a for cydoheximide is increasedS; of translationalfidelity and their effect on other ribosomal properties seemed to indicate • the level of ribosomal subunits in cellthat their products are ribosomal proteins. However, recent sequence data dem- free extracts is elevated4,5; onstrated that one of them (sup2) is highly homologous to an elongation factor gene. • biogenesis of ribosomal subunits is What is the function of the polypeptide products coded for by these genes and the affected s. These data seemed to point to riboreasons for these contradictions? somal proteins as the polypeptide products of the suppressor genes. However, Persistently puzzling suppressors disruption is lethal. The properties of all attempts to find a mutant ~bosomal Recessive suppressors of nonsense co- recessive suppressors s dearly suggest protein with an altered electrophoretic dons in yeast Saccharomyces cerevisiae that both supl and sup2 genes do not mobility by two-dimensional elecwere first described about 25 years agoL code for tRNA, as dominant suppressors trophoretic techniques were unsuccessThese suppressors, designated supl and do, but rather for proteins participating fuP except for one report concerning the sup21.2, or sup35 and sup453, were found in the process of translation. Genetic sup35 (supl) gene6. Moreover, some to be effective towards all three types of characteristics of the recessive suppres- properties of the suppressor genes were nonsense codons (UAA, UAG and sors (e.g. the spectrum of nonsense sup- inconsistent with the conclusion that U G A ) 4'5. They are therefore called 'om- pression, interaction with dominant, and their products are canonical ribosomal nipotent' suppressors, supl is located on tRNA-mediated suppressors) led to the proteins: chromosome II, whereas sup2 is on proposal that they may code for termina- • the size of the suppressor genes coding chromosome IV (Ref. 5). Both genes are tion factors of protein synthesis or pro- region is longer7-9 than all known yeast essential for cell growth and inactivation teins responsible for termination factors ribosomal protein genes; of either of them by mutation or gene binding to the ribosomes 2. • the rate of transcription for supl at The first clues as to how these suppres- least is significantly lower than for ribosomal protein genesg; A. P. Surguchov is at the Institute of Experimental sors work were discovered when analysis Cardiology, USSR Cardiology Research Center, of cell-free translation systems prepared • the codon usage for the suppressor 3rd Cherepkovskaya Street 15,4, Moscow 121552, from these strains showed elevated mis- genes differs from that of ribosomal proUSSR. reading of mRNA compared with the tein genes7.10.
'Omnipotent' nonsense suppressors:
~) I088, Elsevier Publications Cambridge 0376- 5067/88/$02.00
TIBS 13-April 1988
Journal Club A site-specific mRPIA-cytoplasmic protein complex in v/tro Ruth/vL Starzyk Site specific mRNA-protein complexes found in all known ferfitin H and L subregulate the expression of some genes at unit genes, makes up the loop and adjathe level of translation. Among the best- cent stem nucleotides (Fig. 1). Deletion studied systems to exhibit such transla- of the 28-base region eliminates iron tional regulation are prokaryotic regulation of chimeric ferritin-chlorribosomal proteins z. Some of these amphenicoi acetyltransferase or ferdtinoperons are controlled by a translational human growth hormone constructs in feedback mechanism. Here, one of the vivo3,4. Moreover, a synthetic deoxypolypepfide products selectively binds oligonucleotide with this sequence conthe mRNA to prevent further synthesis fers iron responsiveness upon a construct of some or all of the operon's proteins. otherwise unable to respond to iron4. Leibold and Munro 5 incubated cytoLimited sequence specificity and its resulting secondary structure provide the plasmic extracts from rat tissues or cultured rat hepatoma cells with a labeled target site on the mRNA molecule. In higher organisms, translational transcript of part of the 5' untranslated regulation is exemplified by iron- region of rat ferritin L-mRNA, which mediated control of ferritin, an iron- included the 28-base region. Two RNAstorage protein2. In tl:is case, a highly protein complexes, called B1 and B2, conserved 2g-base sequence in the 5' were resolved electrophoretically on untranslated region of ferritin mRNA non-denaturing polyacrylamide gels with has been implicated as a target site for the use of RNase T1 and heparin. Similar the iron-sensitive factor in vivo 3,4. results were obtained with the 5' Recently, Leibold and Munro have iden- untranslated region of rat ferritin Htified a specific cytoplasmic protein mRNA. Competition studies indicate which binds this sequence in vino s. Tiffs that the complexes are specific to the ferprotein-mRNA complex, which is ritin mRNAs. This specificity probably affected by iron, may play a role in the lies in the 28-base sequence because this iron-mediated translational regulation of sequence alone is shared by the 5' untranslated regions of rat ferritin L- and ferritin. Ferritin is found in animal, plant, fun- H-mRNAsS,7. Complex formation in response to iron gal and bacterial cells2. In vertebrates, its protein shell is composed of two types of parallels the translational activation of H subunit: heavy (H) and light (L). Upon and L mRNAs. With liver extracts from addition of iron, synthesis of both sub- rats injected with iron, B2 first units is directed by mRNAs recruited diminishes as the stored mRNA shifts to from a formerly inactive cytoplasmic the polysomes, then disappears entirely, message pool (Ref. 2, and references in and subsequently reappears as the ferriRef. 5). Genes for both subunits have tin mRNA moves back to the RNP parbeen sequenced from a number of ticles. The amount of B1, which starts eukaryotes (see references in Ref. 5). out somewhat greater than that of B2, Computer predictions suggest that the does not diminish over 16 hours. Similar first 76 nucleotides of the rather long fer- results are obtained with heart tissue. ritin 5' untranslated regions can form as With cultured rat hepatoma cells, B2 stem-loop structures. One such com- starts out higher than B 1. As with the tisputer-modeled structure is shown in Fig. sues, though, iron administration effects 1 for the rat ferritin-L mRNA 3,6. The a decrease in B2 with, interestingly, a aforementioned 28-base sequence, concomitant gain in B1. Any reciprocal relationship between B1 and B2 cannot be ascertained from the present data, R. M. Starzyk is at the Deparanent of Biology, Massachusetts Institute of Technology, Cambridge, however. In both complexes the protected RNA MA 02139, USA.
is approximately 50 bases in length. To determine the sequence, cytoplasmic extracts were chosen to produce either B1 alone (iron treatment) or both complexes, and the protected RNA was mapped with RNase T1. In. Fig. l, the protected region, which appears to be the same for both complexes, is boxed. The conserved 28-base sequence (bold letters) is completely protected and lies well within the boundaries of the 50-base area. Leibold and Munro note that at the 3' boundary the protected area might
RGU6 0 R-IJ R-U C-6 U 6 U-R 6-C U 6 C-6 U-R A
,~-- 61 (- 140)
B
U -A G-
['e-
C C C
C- G
G
C- G G A C 16 --I~ (-105)
...AA- U G -- C
A G5' - ~ - - - - - -
(-190) 1 !
GU -
C C 76 (- 125)
i~g. 1. Computer-modeled secondary s~cture of a portion of the ratferritin-L mRNA 5' untranslated region. The conserved 28-base sequence is in bold type. The region protected in the protein-mRNA complexes is boxeds. Numbers indicate positions in the sequence starting from the fu~t transcribed base6. Numbers in parentheses indicate the number of bases 5' to the AUG startcodon6. This figure is after Fig. 1 of Ref. 3 and Fig. 3 of Ref. 6. ~ ) 1088, ~lsevier Publications Cambridge 0376 - 5067/8.8/$02.00
TIBS 13-April 1988
120 extend through four additional unlabeled bases to bring the collective size of the mapped fragments up to that observed for the intact protected region. To identify binding proteins, the labeled complexes were UV crosslinked and subsequently analysed by SDS gel electrophoresis. A single complex of 100 kDa was obtained. Mowing for bound RNA, this would correspond to a protein of about 85 kDa. Proteinase K treatment prior to UV crosslinking prevented complex formation, as did excess unlabeled 5' L-mRNA. Excess unlabeled, nonspecific, competitor RNA did not affect the complex. Iron administration to rats did not change the amount of crosslinked complex formed with tissue extracts. Upon iron addition to cultured cells, however, the crosslinked complex increased in parallel with B1. This suggests that the crosslinked protein
may be a component of B1. In B2, the spatial orientation of the binding protein(s) to the RNA may not favor crosslinking. This work, then, provides the first indication in vitro that iron-sensitive cytoplasmic protein(s) may bind ferritin mRNAs at a specific site and thus regulate their translation. The most likely target is a 28-base sequence highly conserved among all known ferritin mRNAs. In the rat L-mRNA, it is located 142-169 nucleotides before the AUG start of the polypeptide subunit. This region can be drawn in a stem and loop structure as has been suggested for mRNAs of some translationallycontrolled prokaryotic genes (see Fig. 1). At present, however, there is no experimental evidence that such a structure exists. Future work on this system should elucidate the mechanism of iron-
mediated translational control of ferritin synthesis and a role, if any, for site specific mRNA-protein complexes.
References 1 Nomura, M., Gourse, R. and Baughman, G. (1984) Annu. Rev. Biochem. 53, 75-117 2 Theft, E. C. (1987)Annu. Rev. Biochem. 56, 289-315 3 Aziz, N. and Munro, H. N. (1987) Proc. Natl Acad. Sci. USA 84, 8478-8482 4 Hentze, M. W., Caughman, S. W., Rouault, T. A., Barriocanal, J. G., Dancis, A., Harford, J. B. and Klausner, R. D. (1987) Science 238, 1570-1573 5 Leibold, E. A. and Munro, H. N. (1988) Proc. Natl Acad. Sci. USA 85, 2171-2175 6 Leibold, E. A. and Munro, H. N. (1987) J. Biol. Chem. 262, 7335-7341 7 Murray, M. T., White, K. and Munro, H. N. (1987) Proc. Natl Acad. Sci. USA 84, 74387442
Features parent strains~. However, attempts to identify the suppressor gene products gave conflicting results. On the one hand, several properties of ribosomes in the strains carrying recessive suppressor new clues to an old puzzle mutations were found to be changed: • the level of ribosomal ambiguity is AncireiP. Surguchov increased4-~; • the interaction of ribosomes with antiGenetic characterization of omnipotent nonsense-suppressors sup/and sup2 in yeast biotics is altered, e.g. association conoriginally suggested that they code for termination factors, but their role in the control stant K a for cydoheximide is increasedS; of translationalfidelity and their effect on other ribosomal properties seemed to indicate • the level of ribosomal subunits in cellthat their products are ribosomal proteins. However, recent sequence data dem- free extracts is elevated4,5; onstrated that one of them (sup2) is highly homologous to an elongation factor gene. • biogenesis of ribosomal subunits is What is the function of the polypeptide products coded for by these genes and the affected s. These data seemed to point to riboreasons for these contradictions? somal proteins as the polypeptide products of the suppressor genes. However, Persistently puzzling suppressors disruption is lethal. The properties of all attempts to find a mutant ~bosomal Recessive suppressors of nonsense co- recessive suppressors s dearly suggest protein with an altered electrophoretic dons in yeast Saccharomyces cerevisiae that both supl and sup2 genes do not mobility by two-dimensional elecwere first described about 25 years agoL code for tRNA, as dominant suppressors trophoretic techniques were unsuccessThese suppressors, designated supl and do, but rather for proteins participating fuP except for one report concerning the sup21.2, or sup35 and sup453, were found in the process of translation. Genetic sup35 (supl) gene6. Moreover, some to be effective towards all three types of characteristics of the recessive suppres- properties of the suppressor genes were nonsense codons (UAA, UAG and sors (e.g. the spectrum of nonsense sup- inconsistent with the conclusion that U G A ) 4'5. They are therefore called 'om- pression, interaction with dominant, and their products are canonical ribosomal nipotent' suppressors, supl is located on tRNA-mediated suppressors) led to the proteins: chromosome II, whereas sup2 is on proposal that they may code for termina- • the size of the suppressor genes coding chromosome IV (Ref. 5). Both genes are tion factors of protein synthesis or pro- region is longer7-9 than all known yeast essential for cell growth and inactivation teins responsible for termination factors ribosomal protein genes; of either of them by mutation or gene binding to the ribosomes 2. • the rate of transcription for supl at The first clues as to how these suppres- least is significantly lower than for ribosomal protein genesg; A. P. Surguchov is at the Institute of Experimental sors work were discovered when analysis Cardiology, USSR Cardiology Research Center, of cell-free translation systems prepared • the codon usage for the suppressor 3rd Cherepkovskaya Street 15,4, Moscow 121552, from these strains showed elevated mis- genes differs from that of ribosomal proUSSR. reading of mRNA compared with the tein genes7.10.
'Omnipotent' nonsense suppressors:
~) I088, Elsevier Publications Cambridge 0376- 5067/88/$02.00