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Map of cis-acting sequences that determine alternative pre-mRNA processing in the E3 complex transcription unit of
H A Brady1, A Scaria, W S Wold
1Institute for Molecular Virology, St. Louis University School of Medicine, Missouri 63110.
Abstract:
The E3 complex transcription unit of adenovirus encodes four major mRNAs (a, c, f, and h) and two minor (d and e) mRNAs with overlapping exons, alternative splice sites, and two polyadenylation sites, termed E3A (upstream) and E3B (downstream). mRNAs a and d use the E3A polyadenylation site, and mRNAs c, e, f, and h use the E3B site. We have analyzed virus mutants with deletions throughout the E3 region in order to identify cis-acting sequences that function in E3 pre-mRNA processing. The results presented in this report as well as previous results are summarized as follows. (i) Deletions in the first (5') intron at nucleotides (nt) 372 to 768 in E3 had no effect unless they removed the consensus sequence for the nt 372 5' splice site; however, the overall pattern of E3 mRNAs did not change significantly. (ii) Deletions in region I (nt 1441 to 2044) eliminated mRNAs a and c and resulted in corresponding increases in mRNAs f and h; we propose that region I contains sequences that suppress splicing. (iii) Mutations in region II (nt 2161 to 2243) resulted in nearly exclusive synthesis of mRNA f; this phenotype is understood and is discussed. (iv) Changing the AUUAAA component of the E3A poly(A) addition signal to AAUAAA resulted in increased mRNA a levels, suggesting that the E3A poly(A) addition signal is intrinsically inefficient. (v) Deletions in region III (nt 2488 to 3002) decreased mRNA a levels about two- to threefold and specifically increased mRNA f levels; we suggest that region III facilitates use of the E3A polyadenylation site. (vi) Deletions in region IV (nt 2904 to 3251) increased mRNA a levels about two- to threefold; we suggest that region IV may contain sequences that facilitate use of the E3B polyadenylation site. A map of sequences that determine alternative pre-mRNA processing in region E3 is now nearly complete.
Insights
Adenovirus E3 pre-mRNA processing involves complex alternative splicing. Researchers identified specific DNA regions regulating splice site selection and polyadenylation, creating a map of cis-acting sequences for E3 gene expression.
Area of Science:
- Molecular Biology
- Virology
- Gene Regulation
Background:
- The adenovirus E3 transcription unit produces multiple mRNAs (a, c, f, h, d, e) through alternative splicing.
- These mRNAs utilize two distinct polyadenylation sites: E3A (upstream) and E3B (downstream).
- Understanding the cis-acting sequences governing E3 pre-mRNA processing is crucial for deciphering viral gene expression.
Purpose of the Study:
- To identify cis-acting sequences involved in adenovirus E3 pre-mRNA processing.
- To map regulatory elements controlling alternative splicing and polyadenylation within the E3 region.
Main Methods:
- Analysis of adenovirus mutants with deletions across the E3 transcription unit.
- Characterization of mRNA profiles resulting from these mutations.
- Identification of specific nucleotide regions (I, II, III, IV) and splice sites affecting mRNA production.
Main Results:
- Deletions in Region I suppressed splicing, favoring mRNAs f and h over a and c.
- Mutations in Region II led to predominant synthesis of mRNA f.
- Alterations in the E3A polyadenylation signal and Regions III/IV modulated the usage of E3A and E3B polyadenylation sites, impacting mRNA a and f levels.
Conclusions:
- Specific DNA sequences within the adenovirus E3 region act in cis to regulate alternative pre-mRNA processing.
- A comprehensive map of these regulatory sequences is nearing completion.
- This research provides critical insights into the mechanisms controlling viral mRNA production and alternative splicing.