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Determination of 3′ end processing in retroelements
OMMENT
inhibitors or by mutations in genes required for DNA synthesis prevents mitosis from taking place. In S. cerevisiae, the RAD9 gene has been reported to be a component of the system that checks the completion of DNA replication before mitosis is permitted to take place n. Temperature-sensitive mutants defective in most DNA replication functions do not undergo mitosis at the restrictive temperature. However, if these mutants also carry a rad9 gene defect, cells continue through an aberrant mitosis into the next cell cycle. This has catastrophic consequences, including a dramatic loss of cell viability. The recent work of Elledge and Davis9 has also revealed a RAD,9independent checkpoint for regulation of the cell cycle. Mutations in the RNR1 or RNR2 genes, as well as hydroxyurea (which is a specific inhibitor of RNR), arrest cells with a terminal phenotype that is independent of the RAD9 gene. Elledge and Davis suggest that the control pathway that responds to arrest by hydroxyurea is more likely to be the one involved in the
coordination of DNA synthesis and mitosis. They predict that any cells arrested with a cdc phenoty,pe due to a block of DNA replication should be RAD9 independent, and this prediction is supported by the behaviour of cdc8 in a mutant rad9 background lz. The CDC8 gene product, thymidylate kinase, is involved in the formation of precursors required for DNA synthesis, as is RNR. This might distinguish these two genes from those required for DNA chain elongation, defects in which are sensed by RAD9. It is possible that the two different aspects of DNA metabolism are detected by different pathways, and this suggests the existence of two checkpoints acting to delay mitosis until DNA synthesis is completed. If two types of control do exist, other genes involved in the synthesis of precursors, such as CDC21 (encoding thymidylate synthase), should also show RAD9 independence. A search for mutations analogous to rad9 that allow RNR or CDC8 mutants, or hydroxyurea-
arrested cells, to undergo mitosis could confirm the existence of the proposed hydroxyurea checkpoint in the cell.
References 1 Elledge, s.J. and Davis, R.W. (1987) Mol. Cell. Biol. 7, 2783-2793 2 Bjorklund, S., Skog, S., Tribukait, B. and Thelander, L. Biochemistry (in press) 3 Thelander, L. and Berg, P. (1986) Mol. Cell. Biol. 6, 3433-3442 4 Gordon, C.B. and Fantes, P.A. (1986) EMBOJ. 5, 2981-2985 5 Elledge, S.J. and Davis, R.W. (1989) Mol. Cell. Biol. 9, 4932--4940 6 Elledge, S.J. and Davis, R.W. (1989) MoL Cell. Biol. 9, 5373-5386 7 Hurd, H. and Roberts, J. (1989) Mol. Cell. Biol. 9, 5361-5372 8 Hurd, H., Roberts, C.W. and Roberts, J.W. (1987) Mol. Cell. Biol. 7, 3673-3677 .9 Elledge, S.J. and Davis, R.W. (1990) Genes Dev. 4, 740-751 10 Thelander, M. and Thelander, L. (1989) FAIBOJ. 8, 2475-2479 11 Hartwell, L.H. and Weinert, T.A. (1989) Science246, 629-634 12 Schiestl, R.H., Reynolds, P., Prakash, S. and Prakash, L. (1989) Mol. Cell. Biol. 9, 1882-1896
Determination of 3' end processing in retr0elements JOHN M. COFFIN AND CLAIREMOORE DEPARTMENTOFMOLECULARBIOLOGYANDMICROBIOLOGY, TUFTS UNIVERSITY SCHOOL OF
MEDICINE,
136 HARRISONAVENUE,BOSTON,MA02111, USA. Retroviruses and transposable elements that replicate via reverse transcription of RNA into DNA and then transcribe the DNA copy into RNA face a special problem in their replication. The RNA transcript must be convertible to a transcription unit, so the RNA must contain the signals necessary to direct transcription initiation and RNA processing by cellular systems. In particular, eukaryotic transcription units must contain elements upstream of the transcribed region comprising binding sites for RNA polymerase II and for factors enhancing and regulating transcriptional initiation. Sequences downstream of the final RNA product contribute to cleavage of a larger precursor to form the RNA 3' end. In retroviruses, signals such as
the above, which can only function when they lie outside the final RNA product, must also be encoded within the viral RNA. Retroviruses solve this problem by employing a process of reverse transcription that duplicates terminal RNA sequences to both ends of the DNA molecule (Fig. 1). The structures thus created are known as long terminal repeats (LTRs) and contain most, if not all, c/s-acting regulatory elements for transcription and polyadenylation. Hepadnaviruses such as hepatitis B virus accomplish a similar end by the use of a single set of sequences, but in the context of a closed circular DNA molecule. The synthesis of these structures solves the problems of providing upstrea~ downstream signals, but c r e ~ n t h e r potential TIG SEPTEMBER1990 VOL.6 NO. 9
©i990 Elsevier Science Publishers Lid ¢UK) 0168 - 9479/90/$02.00
problem: the inappropriate juxtaposition of these signals in the DNA and primary transcript. This problem is made particularly acute by the necessity, for the purpose of aligning the jumps during reverse transcription, of creating a terminally redundant sequence in the RNAx. Thus, some of the same sequences are transcribed twice and appear once near the 3' end and once near the 5' end of the RNA (Fig. 1). Since the sequences important for specifying polyadenylation are near the 3' end of the mRNA, they will also often be found near the 5' end. What then prevents the premature processing of transcripts using the 5' copy of the signals? With some retroviruses, the solution is obvious: the redundant sequence is so short that the 5' copy
OMMENT of the AATAAA polyadenylation signal is not found in the tranRetrovirus scrip#. With others, such as human T-lymphotropic virus, the poly(A) signal precedes the site of cleavage Ill ill] and polyadenylation by nearly 300 bases, and lies about 50 bases upstream of the initiation site for nn tIl transcription. The correct relationship to the site of 3' processing is apparently maintained by an unusually stable secondary structure comprising most of the intervening RNA. With many other retroviruses, as well as hepadnaviruses, the known c~acting signals for polyadenylation lie within the terminally redundant sequence near either end. What blocks the use of the 5'most signal and the generation of severely truncated transcripts? A study recently reported by Russnak and Ganem3 addresses this issue for hepatitis B virus. These authors noted that the polyadenylation signal of two different hepadnaviruses is apparently TATAAA rather than the canonical (and presumably FIGH optimal) AATAAA. To test the roles Localizations of signals for pol~adeny!ation.The transcription unit of a retrovirus (top) of this signal and surrounding se- and hepatitis B virus (HBV; bottom) are shown with the primary transcription and quences, different amounts of hep- polyadenylated genome (or pregenome in the case of HBV). The solid region indicates atitis DNA were placed between the AATAApoly(A) signal; the stippled box shows the appropriate location of the the SV40 early promoter and the region identified by Russnak and Ganem3 as necessary for efficient polyadenylation. SV40 late polyadenylation site. After introduction of these con- the U3 region from the LTRs of two that the regulatory sequence is structs into cells, the efficiency of unrelated retroviruses - spleen highly degenerate, or that multiple polyadenylation at the hepatitis necrosis virus and human immuno- mechanisms are responsible for the virus site was compared to that at deficiency virus - substituted for effect. How these sequences (or the hepatitis upstream sequence in factors that interact with them) the downstream SV40 site. The results clearly showed that an orientation-dependent manner. influence the normal polyadenylif the construct contained 107 bp of Others have also shown that ation machinery remains, enticingly, hepatitis sequence upstream of its sequences in the U3 region are an open issue. poly(A) site, very little polyadenyl- required for efficient use of retroation occurred at this site, analo- viral polyadenylation site#. In fact, gous to the 'f'wst pass' through the this enhancement is not restricted References 1 Varmus, H.E. and Swanstrom, R. signal after initiation. Perhaps not to retroelements, since the positive (1982) in RNA Tumor Viruses (2nd surprisingly, exchanging TATAAA influence of sequences upstream of edn, part 1) (Weiss, R., Teich, N., for AATAAA permitted efficient first- the AATAAA has been demonstrated Varmus, H. and Coffin, J., eds), pp. pass processing at the hepatitis site. for the SV40 late 5, the adenovirus L1 369-512, Cold Spring Harbor However, inclusion of an additional (Ref. 6), and the cauliflower mosaic Laboratory Press 290 bp of hepatitis sequence virus (B. Mogan et aL, submitted) 2 Coffin,J.M. (1985) in RNA Tumor Viruses(2nd edn, part 1) (Weiss, R.. upstream of the hepatitis poly(A) polyadenylation sites. It will be Teich, N., Varmus, H. and Coffin, J., site was also sufficient to restore interesting to see if the processing eds), pp. 17-74, Cold Spring nearly full processing of that site. of any cellular transcripts is affected Harbor Laboratory Press Thus, a region near but not at the in a similar manner, and whether 3 Russnak, R. and Ganem, D. (1990) modulating the efficiency of polyo 3' end of the hepatitis RNA (and Genes Dev. 4, 764-776 missing from the 5' end) specified adenylation through such sequences 4 Dougherty, J.P. and Temin, H.M.. 3' end formation by enhancing the becomes yet another means of con(1987) Proc. Natl Acad. Sci. USA 84. activity of an otherwise inefficient trolling gene expression. 1197-1201 Analysis of primary sequence or 5 Carswell, S. and Alwine, J.C. (1989) polyadenylation signal. Is this a general mechanism secondary structure has not reMol. Cell. Biol. 9, 4248-4258 6 DeZazzo, J.D. and Imperiale, H.M. applicable to other retroelements? vealed any similarities among these (1978) Mol. Cell. Biol. 9, 4951-4961 It may be, since, in the same assay, upstream regions, indicating either
I
"lag SEPTEMBER1990 VOL.6 NO. 9
m
II
I
inhibitors or by mutations in genes required for DNA synthesis prevents mitosis from taking place. In S. cerevisiae, the RAD9 gene has been reported to be a component of the system that checks the completion of DNA replication before mitosis is permitted to take place n. Temperature-sensitive mutants defective in most DNA replication functions do not undergo mitosis at the restrictive temperature. However, if these mutants also carry a rad9 gene defect, cells continue through an aberrant mitosis into the next cell cycle. This has catastrophic consequences, including a dramatic loss of cell viability. The recent work of Elledge and Davis9 has also revealed a RAD,9independent checkpoint for regulation of the cell cycle. Mutations in the RNR1 or RNR2 genes, as well as hydroxyurea (which is a specific inhibitor of RNR), arrest cells with a terminal phenotype that is independent of the RAD9 gene. Elledge and Davis suggest that the control pathway that responds to arrest by hydroxyurea is more likely to be the one involved in the
coordination of DNA synthesis and mitosis. They predict that any cells arrested with a cdc phenoty,pe due to a block of DNA replication should be RAD9 independent, and this prediction is supported by the behaviour of cdc8 in a mutant rad9 background lz. The CDC8 gene product, thymidylate kinase, is involved in the formation of precursors required for DNA synthesis, as is RNR. This might distinguish these two genes from those required for DNA chain elongation, defects in which are sensed by RAD9. It is possible that the two different aspects of DNA metabolism are detected by different pathways, and this suggests the existence of two checkpoints acting to delay mitosis until DNA synthesis is completed. If two types of control do exist, other genes involved in the synthesis of precursors, such as CDC21 (encoding thymidylate synthase), should also show RAD9 independence. A search for mutations analogous to rad9 that allow RNR or CDC8 mutants, or hydroxyurea-
arrested cells, to undergo mitosis could confirm the existence of the proposed hydroxyurea checkpoint in the cell.
References 1 Elledge, s.J. and Davis, R.W. (1987) Mol. Cell. Biol. 7, 2783-2793 2 Bjorklund, S., Skog, S., Tribukait, B. and Thelander, L. Biochemistry (in press) 3 Thelander, L. and Berg, P. (1986) Mol. Cell. Biol. 6, 3433-3442 4 Gordon, C.B. and Fantes, P.A. (1986) EMBOJ. 5, 2981-2985 5 Elledge, S.J. and Davis, R.W. (1989) Mol. Cell. Biol. 9, 4932--4940 6 Elledge, S.J. and Davis, R.W. (1989) MoL Cell. Biol. 9, 5373-5386 7 Hurd, H. and Roberts, J. (1989) Mol. Cell. Biol. 9, 5361-5372 8 Hurd, H., Roberts, C.W. and Roberts, J.W. (1987) Mol. Cell. Biol. 7, 3673-3677 .9 Elledge, S.J. and Davis, R.W. (1990) Genes Dev. 4, 740-751 10 Thelander, M. and Thelander, L. (1989) FAIBOJ. 8, 2475-2479 11 Hartwell, L.H. and Weinert, T.A. (1989) Science246, 629-634 12 Schiestl, R.H., Reynolds, P., Prakash, S. and Prakash, L. (1989) Mol. Cell. Biol. 9, 1882-1896
Determination of 3' end processing in retr0elements JOHN M. COFFIN AND CLAIREMOORE DEPARTMENTOFMOLECULARBIOLOGYANDMICROBIOLOGY, TUFTS UNIVERSITY SCHOOL OF
MEDICINE,
136 HARRISONAVENUE,BOSTON,MA02111, USA. Retroviruses and transposable elements that replicate via reverse transcription of RNA into DNA and then transcribe the DNA copy into RNA face a special problem in their replication. The RNA transcript must be convertible to a transcription unit, so the RNA must contain the signals necessary to direct transcription initiation and RNA processing by cellular systems. In particular, eukaryotic transcription units must contain elements upstream of the transcribed region comprising binding sites for RNA polymerase II and for factors enhancing and regulating transcriptional initiation. Sequences downstream of the final RNA product contribute to cleavage of a larger precursor to form the RNA 3' end. In retroviruses, signals such as
the above, which can only function when they lie outside the final RNA product, must also be encoded within the viral RNA. Retroviruses solve this problem by employing a process of reverse transcription that duplicates terminal RNA sequences to both ends of the DNA molecule (Fig. 1). The structures thus created are known as long terminal repeats (LTRs) and contain most, if not all, c/s-acting regulatory elements for transcription and polyadenylation. Hepadnaviruses such as hepatitis B virus accomplish a similar end by the use of a single set of sequences, but in the context of a closed circular DNA molecule. The synthesis of these structures solves the problems of providing upstrea~ downstream signals, but c r e ~ n t h e r potential TIG SEPTEMBER1990 VOL.6 NO. 9
©i990 Elsevier Science Publishers Lid ¢UK) 0168 - 9479/90/$02.00
problem: the inappropriate juxtaposition of these signals in the DNA and primary transcript. This problem is made particularly acute by the necessity, for the purpose of aligning the jumps during reverse transcription, of creating a terminally redundant sequence in the RNAx. Thus, some of the same sequences are transcribed twice and appear once near the 3' end and once near the 5' end of the RNA (Fig. 1). Since the sequences important for specifying polyadenylation are near the 3' end of the mRNA, they will also often be found near the 5' end. What then prevents the premature processing of transcripts using the 5' copy of the signals? With some retroviruses, the solution is obvious: the redundant sequence is so short that the 5' copy
OMMENT of the AATAAA polyadenylation signal is not found in the tranRetrovirus scrip#. With others, such as human T-lymphotropic virus, the poly(A) signal precedes the site of cleavage Ill ill] and polyadenylation by nearly 300 bases, and lies about 50 bases upstream of the initiation site for nn tIl transcription. The correct relationship to the site of 3' processing is apparently maintained by an unusually stable secondary structure comprising most of the intervening RNA. With many other retroviruses, as well as hepadnaviruses, the known c~acting signals for polyadenylation lie within the terminally redundant sequence near either end. What blocks the use of the 5'most signal and the generation of severely truncated transcripts? A study recently reported by Russnak and Ganem3 addresses this issue for hepatitis B virus. These authors noted that the polyadenylation signal of two different hepadnaviruses is apparently TATAAA rather than the canonical (and presumably FIGH optimal) AATAAA. To test the roles Localizations of signals for pol~adeny!ation.The transcription unit of a retrovirus (top) of this signal and surrounding se- and hepatitis B virus (HBV; bottom) are shown with the primary transcription and quences, different amounts of hep- polyadenylated genome (or pregenome in the case of HBV). The solid region indicates atitis DNA were placed between the AATAApoly(A) signal; the stippled box shows the appropriate location of the the SV40 early promoter and the region identified by Russnak and Ganem3 as necessary for efficient polyadenylation. SV40 late polyadenylation site. After introduction of these con- the U3 region from the LTRs of two that the regulatory sequence is structs into cells, the efficiency of unrelated retroviruses - spleen highly degenerate, or that multiple polyadenylation at the hepatitis necrosis virus and human immuno- mechanisms are responsible for the virus site was compared to that at deficiency virus - substituted for effect. How these sequences (or the hepatitis upstream sequence in factors that interact with them) the downstream SV40 site. The results clearly showed that an orientation-dependent manner. influence the normal polyadenylif the construct contained 107 bp of Others have also shown that ation machinery remains, enticingly, hepatitis sequence upstream of its sequences in the U3 region are an open issue. poly(A) site, very little polyadenyl- required for efficient use of retroation occurred at this site, analo- viral polyadenylation site#. In fact, gous to the 'f'wst pass' through the this enhancement is not restricted References 1 Varmus, H.E. and Swanstrom, R. signal after initiation. Perhaps not to retroelements, since the positive (1982) in RNA Tumor Viruses (2nd surprisingly, exchanging TATAAA influence of sequences upstream of edn, part 1) (Weiss, R., Teich, N., for AATAAA permitted efficient first- the AATAAA has been demonstrated Varmus, H. and Coffin, J., eds), pp. pass processing at the hepatitis site. for the SV40 late 5, the adenovirus L1 369-512, Cold Spring Harbor However, inclusion of an additional (Ref. 6), and the cauliflower mosaic Laboratory Press 290 bp of hepatitis sequence virus (B. Mogan et aL, submitted) 2 Coffin,J.M. (1985) in RNA Tumor Viruses(2nd edn, part 1) (Weiss, R.. upstream of the hepatitis poly(A) polyadenylation sites. It will be Teich, N., Varmus, H. and Coffin, J., site was also sufficient to restore interesting to see if the processing eds), pp. 17-74, Cold Spring nearly full processing of that site. of any cellular transcripts is affected Harbor Laboratory Press Thus, a region near but not at the in a similar manner, and whether 3 Russnak, R. and Ganem, D. (1990) modulating the efficiency of polyo 3' end of the hepatitis RNA (and Genes Dev. 4, 764-776 missing from the 5' end) specified adenylation through such sequences 4 Dougherty, J.P. and Temin, H.M.. 3' end formation by enhancing the becomes yet another means of con(1987) Proc. Natl Acad. Sci. USA 84. activity of an otherwise inefficient trolling gene expression. 1197-1201 Analysis of primary sequence or 5 Carswell, S. and Alwine, J.C. (1989) polyadenylation signal. Is this a general mechanism secondary structure has not reMol. Cell. Biol. 9, 4248-4258 6 DeZazzo, J.D. and Imperiale, H.M. applicable to other retroelements? vealed any similarities among these (1978) Mol. Cell. Biol. 9, 4951-4961 It may be, since, in the same assay, upstream regions, indicating either
I
"lag SEPTEMBER1990 VOL.6 NO. 9
m
II
I