Elements upstream of the AAUAAA within the human immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in vitro.
Open accessAbstract
Recent in vivo studies have identified specific sequences between 56 and 93 nucleotides upstream of a polyadenylation [poly(A)] consensus sequence, AAUAAA, in human immunodeficiency virus type 1 (HIV-1) that affect the efficiency of 3'-end processing at this site (A. Valsamakis, S. Zeichner, S. Carswell, and J. C. Alwine, Proc. Natl. Acad. Sci. USA 88:2108-2112, 1991). We have used HeLa cell nuclear extracts and precursor RNAs bearing the HIV-1 poly(A) signal to study the role of upstream sequences in vitro. Precursor RNAs containing the HIV-1 AAUAAA and necessary upstream (U3 region) and downstream (U5 region) sequences directed accurate cleavage and polyadenylation in vitro. The in vitro requirement for upstream sequences was demonstrated by using deletion and linker substitution mutations. The data showed that sequences between 56 and 93 nucleotides upstream of AAUAAA, which were required for efficient polyadenylation in vivo, were also required for efficient cleavage and polyadenylation in vitro. This is the first demonstration of the function of upstream sequences in vitro. Previous in vivo studies suggested that efficient polyadenylation at the HIV-1 poly(A) signal requires a spacing of at least 250 nucleotides between the 5' cap site and the AAUAAA. Our in vitro analyses indicated that a precursor containing the defined upstream and downstream sequences was efficiently cleaved at the polyadenylation site when the distance between the 5' cap and the AAUAAA was reduced to at least 140 nucleotides, which is less than the distance predicted from in vivo studies. This cleavage was dependent on the presence of the upstream element.
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Specific sequences in the U3 region upstream of the conserved hexamer boost polyadenylation by improving CPSF binding both in vitro and in vivo.
“These sequences have been shown to boost polyadenylation by improving CPSF binding both in vitro as well as in vivo (Brown et al. 1991, Valsamakis et al. 1992, Ashe et al. 1995, Gilmartin et al. 1995, Graveley and Gilmartin 1996).”
From Beyond reverse transcription: molecular mechanisms and emerging paradigms in retroviral replication (2026) · cites this paper as Valsamakis et al. 1992
The Discussion (page not provided) says the upstream sequences are required for efficient polyadenylation in vitro and agrees with earlier in vivo results. It describes increased binding of the polyadenylation complex as a likely mechanism, but says the mechanism remains unknown; it does not establish improved CPSF binding. (LLM, conf=0.88)
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References 49 · Cited by 70
Research works printed but not linked: 4
- Ausubel, F. A., R. Brent, R. E. Lingstrom, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1987. Current protocols in molecular biology. Greene Publishing and Wiley- Interscience, New York.
- Conway, L., and M. Wickens. 1985. A sequence downstream of AAUAAA is required for formation of simian virus 40 late mRNA 3' termini in frog oocytes. Proc. Natl. Acad. Sci. USA 82:3949-3953.
- Galli, G., J. Guise, P. W. Tucker, and J. R. Nevins. 1988. Poly(A) site choice rather than splice site choice governs the regulated production of IgM heavy-chain RNAs. Proc. Natl. Acad. Sci. USA 85:2439-2443.
- Valsamakis, A., S. Zeichner, S. Carswell, and J. C. A}wine. 1991. The human immunodeficiency virus type 1 polyadenylation signal: a 3' long terminal repeat element upstream of the AAUAAA necessary for efficient polyadenylation. Proc. Natl. Acad. Sci. USA 88:2108-2112.
- Authors
- Valsamakis, A., Schek, N., & Alwine, J. C.
- Affiliations
- University of Pennsylvania (US)
- ORCID
- Alwine, J C
- Venue
- Molecular and Cellular Biology
- Publisher
- Informa UK Limited
- Published
- 1992-09
- Volume
- 12
- Issue
- 9
- Pages
- 3699-3705
- ISSN
- 0270-7306 (ISSN-L)
- 1098-5549
- PMC
- PMC360226
- OpenAlex
- W2130862231
- Cited by
- 70
- References
- 49
- Citations by year
- 2019: 1 · 2018: 1 · 2017: 1 · 2013: 3 · 2012: 1
- FWCI
- 2.65
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Molecular and Cellular Biology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- University of Pennsylvania
- National Institutes of Health
- National Institute of General Medical Sciences (GM45773, R01GM045773)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- HIV-1, polyadenylation signal, upstream sequences, 3' end processing, U3 region, U5 region, RNA cleavage, RNA precursors
- MeSH terms
- Base Sequence, HeLa Cells, Humans, Molecular Sequence Data, Poly A, Regulatory Sequences, Nucleic Acid, RNA Precursors, RNA Processing, Post-Transcriptional, RNA, Viral, HIV-1, Mutagenesis
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