Poly(A) site selection in the adenovirus type 2 major late transcription unit: Processing and regulatory signals.

Abstract

The adenovirus major late transcription unit (MLTU) encodes five mRNA families, L1 through L5, each distinguished by a unique poly(A) site. Poly(A) site selection is regulated during infection, predominating early at the L1 site and late at the L2-L5 sites. In this thesis, critical cis-acting signals involved in L1 and L2 poly(A) site usage are identified. Through transient expression analysis of plasmids harboring mutated versions of the L2 poly(A) site, an important 3$\sp\prime$ end processing element between 7 and 27 bp downstream of the L2 poly(A) site is identified. Insertions and small deletions within this region significantly lower the efficiency of L2 mRNA 3$\sp\prime$ end formation. Although no single nucleotide in the region is absolutely required for function, the entire element is necessary for maximal 3$\sp\prime$ end processing efficiency. Through a similar analysis of complex transcription units harboring mutated versions of the L1 poly(A) site, a critical 3$\sp\prime$ end processing signal is identified in the sequences upstream of AAUAAA at the L1 poly(A) site. Functioning independent of the major late promoter, but relying on physical linkage of the processing signals, the upstream sequences help govern predominant L1 poly(A) site usage at either position of a complex transcription unit encoding two poly(A) sites and moreover, may confer predominant L1 poly(A) site usage only in the presence of L1 downstream sequences. To study the potential regulatory role of L1 sequences on the adenovirus chromosome, a set of recombinant adenoviruses was generated, each harboring a miniature version of the MLTU encoding the L1 and L3 sites. Through mutagenesis of the L1 sequences in the mini-MLTUs, it is demonstrated that temporal regulation of the L1 poly(A) site relies on cis-acting elements upstream of AAUAAA and downstream of +33. Completely dispensable for efficient 3$\sp\prime$ end processing in simple transcription units, each of these regulatory elements is needed for optimal L1 poly(A) site selection in an early infection and its subsequent down regulation during a late infection.