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Duplication of functional polyadenylation signals in polyomavirus DNA does not alter efficiency of polyadenylation or
Abstract:
We constructed viable insertion mutants of polyomavirus that contain duplications of the nucleotide sequences surrounding the polyadenylation sites for both E- and L-strand RNAs. Our results showed that formation of poly(A)+ 3'termini of L-strand mRNAs requires sequence elements located between 12 and 87 nucleotides downstream of AAUAAA. No more than 19 nucleotides upstream and 44 nucleotides downstream of AAUAAA are required for polyadenylation of E-strand mRNAs. Our results and those of others suggest that there are three distinct sequence elements required for mRNA 3' end formation: AAUAAA and two downstream elements. An insertion mutant containing two adjacent functional polyadenylation signals produced E-strand and L-strand mRNAs with 3' ends at both sites. However, the overall level of polyadenylation of L-strand RNAs was not increased over the low (10 to 25%) levels seen with wild-type virus. Neither was the efficiency of termination of L-strand transcription increased in mutant virus-infected cells. We conclude that factors required for both polyadenylation and transcription termination are limiting in polyomavirus-infected mouse cells.
Insights
Polyomavirus mRNA 3' end formation requires specific DNA sequences downstream of AAUAAA. Factors for polyadenylation and transcription termination appear limited in infected mouse cells.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Polyadenylation is crucial for mRNA stability and translation.
- Understanding mRNA 3' end formation in viruses like polyomavirus provides insights into host-pathogen interactions.
Purpose of the Study:
- To investigate the sequence requirements for polyadenylation of E- and L-strand RNAs in polyomavirus.
- To determine the role of specific downstream elements in mRNA 3' end formation.
Main Methods:
- Construction of viable polyomavirus insertion mutants with duplicated sequences around polyadenylation sites.
- Analysis of polyadenylation efficiency and transcription termination in mutant virus-infected cells.
Main Results:
- Identified specific downstream nucleotide sequences essential for L-strand mRNA polyadenylation (12-87 nucleotides).
- Defined upstream (≤19 nucleotides) and downstream (≤44 nucleotides) requirements for E-strand mRNA polyadenylation.
- Observed no increase in L-strand polyadenylation or transcription termination efficiency despite engineered dual polyadenylation signals.
Conclusions:
- Three distinct sequence elements (AAUAAA and two downstream elements) are necessary for mRNA 3' end formation.
- Factors controlling polyadenylation and transcription termination are limiting in polyomavirus-infected mouse cells.