RNA Polymerase II-dependent Positional Effects on mRNA 3′ End Processing in the Adenovirus Major Late Transcription Unit

Abstract

During the early phase of adenovirus infection, the promoter-proximal L1 poly(A) site in the major late transcription unit is used preferentially despite the fact that the distal L3 poly(A) site is stronger (i.e. it competes better for processing factors and is cleaved at a faster rate, in vitro). Previous work had established that this was due at least in part to the stable binding of the processing factor, cleavage and polyadenylation specificity factor, to the L1 poly(A) site as mediated by specific regulatory sequences. It is now demonstrated that in addition, the L1 poly(A) site has a positional advantage because of its 5 location in the transcription unit. We also show that preferential processing of a particular poly(A) site in a complex transcription unit is dependent on RNA polymerase II. Our results are consistent with recent reports demonstrating that the processing factors cleavage and polyadenylation specificity factor and cleavage stimulatory factor are associated with the RNA polymerase II holoenzyme; thus, processing at a weak poly(A) site like L1 can be enhanced by virtue of its being the first site to be transcribed.

The RNA Tether from the Poly(A) Signal to the Polymerase Mediates Coupling of Transcription to Cleavage and Polyadenylation

“…Although the present study has a number of features in common with previous studies on the coupling of 3 0 end processing to transcription, it is difficult to make direct comparisons. For example, like us, Mifflin and Kellems (1991) observed processing that was fast and efficient when coupled to transcription, and Yonaha and Proudfoot (1999; and Ahuja et al (2001) observed functional interactions between processing and transcription. Nevertheless, it is not possible to say if a tether requirement would have been evident in those studies, because they all employed crowding agents like PVA to enhance the processing.…”

“…Very likely, newly extruded RNA is packaged in a way that makes it a preferred substrate for the next step of mRNA production, but this is really coupling in the broader precursor-product sense. For this reason, studies directed at functional coupling often compare the processing of RNA produced by RNA polymerase II with the processing of RNA produced by T7 RNA polymerase under the same conditions (Mifflin and Kellems, 1991;Ahuja et al, 2001). However, even here caution is required because, as discussed earlier, mere involvement of the polymerase as a participant in the processing reaction is not synonymous with functionally coupling processing to transcription.…”

Transcriptional Blocks Limit Adenoviral Replication in Primary Ovarian Tumor

“…For example, serine-rich proteins within the cell have been shown to suppress viral infection by blocking the obligate splicing of adenoviral pre-mRNA (15). Furthermore, the decrease in adenoviral replication may be due to ineffective or absent cleavage of polyadenylated RNA (16). In both of these cases, specific factors expressed by the cells may influence the outcome of productive infection.…”