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.

Polyadenylation and transcription termination in gene constructs containing multiple tandem polyadenylation signals

“…Transcription termination and polyadenylation are closely linked in vivo (9)(10)(11)(12). RNA polymerase II transcription termination occurs hundreds or thousands of nucleotides downstream of polyadenylation sites (7,13,14).…”

“…RNA polymerase II transcription termination occurs hundreds or thousands of nucleotides downstream of polyadenylation sites (7,13,14). Although there have been a number of reports of specific sequences which may act as termination sequences (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), in vivo, transcription termination often depends on 3'-end processing (9)(10)(11)(12)26). Mutations in the conserved polyadenylation AAUAAA signal of the mouse major 3-globin gene or the human globin gene, respectively (11,12), or mutations in both the AAUAAA hexanucleotide and the downstream GT-rich region of the SV40 early polyadenylation site (9) can inhibit polyadenylation as well as transcription termination.…”

“…Mutations in the conserved polyadenylation AAUAAA signal of the mouse major 3-globin gene or the human globin gene, respectively (11,12), or mutations in both the AAUAAA hexanucleotide and the downstream GT-rich region of the SV40 early polyadenylation site (9) can inhibit polyadenylation as well as transcription termination. Further, insertion of a strong poly(A) signal (such as the rabbit 1-globin poly(A) site) upstream of a weak poly(A) signal (such as the polyomavirus late poly(A) site) can induce efficient transcription termination (10).…”

“…Cytoplasmic RNAs were analyzed by RNase T2 protection experiments as previously described (39). The (10) into the ApaI gap of pRUEY-1.…”

“…Briefly, the polyoma late polyoma's poly(A) signal was replaced with a strong, well-characterized synthetic poly(A) site (50), while preserving the viral early coding region and early poly(A) signal on the opposite DNA strand. Plasmid pa-a was made by inserting a fragment comprising 50 Construct pR-,3G contains the genome of our parent laboratory strain 59RA, but is identical in its poly(A) region to mutant ins5, which has been shown to exhibit efficient late-strand polyadenylation and transcription termination (10). In order to confirm that the rabbit 3-globin poly(A) signal of pR-,BG directs efficient transcription termination relative to our wild type construct, nuclear run-on assays were performed (Fig.…”