A rabbit beta-globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA

Abstract

We constructed a viable insertion mutant (ins 5) of polyomavirus which contains, upstream of the L-strand polyadenylation signal, a 94-nt fragment of rabbit beta-globin DNA. Included in this fragment are all of the sequence elements required for efficient cleavage and polyadenylation of rabbit beta-globin RNA. The beta-globin signal was efficiently recognized by the cleavage/polyadenylation machinery in mouse 3T6 cells infected with ins 5, signalling greater than 90% of the polyadenylation events on L-strand RNAs. Furthermore, the presence of this efficient polyadenylation signal resulted in a 1.4- to 2.5-fold increase in the fraction of virus-specific RNAs that were polyadenylated. Most importantly, termination of transcription by RNA polymerase II on ins 5 DNA was also increased compared with wild-type virus; nearly 100% of polymerases terminated per traverse of the ins 5 genome. These findings demonstrate that the rabbit beta-globin insert, which contains a strong polyadenylation signal, also contains at least part of a signal for termination of transcription by RNA polymerase II. These results also show that the multiple, spliced leaders on polyomavirus L-strand mRNAs, which arise as a result of inefficient termination and polyadenylation, are not necessary for efficient virus replication.