The conserved AAUAAA hexamer of the poly(A) signal can act alone to trigger a stable decrease in RNA polymerase II transcription velocity

Abstract

In vivo the poly(A) signal not only directs 39-end processing but also controls the rate and extent of transcription. Thus, upon crossing the poly(A) signal RNA polymerase II first pauses and then terminates. We show that the G/U-rich region of the poly(A) signal, although required for termination in vivo, is not required for poly(A)-dependent pausing either in vivo or in vitro. Consistent with this, neither CstF, which recognizes the G/U-rich element, nor the polymerase CTD, which binds CstF, is required for pausing. The only part of the poly(A) signal required to direct the polymerase to pause is the AAUAAA hexamer. The effect of the hexamer on the polymerase is long lasting-in many situations polymerases over 1 kb downstream of the hexamer continue to exhibit delayed progress down the template in vivo. The hexamer is the first part of the poly(A) signal to emerge from the polymerase and may play a role independent of the rest of the poly(A) signal in paving the way for subsequent events such as 39-end processing and termination of transcription.

Transcription termination by nuclear RNA polymerases

“…It has been suggested that the poly(A) signal itself can direct RNAPII pausing. The AAUAAA-dependent pausing is mediated by CPSF but does not require the CTD and any other downstream pausing elements (Orozco et al 2002;Park et al 2004;Nag et al 2006Nag et al , 2007. Another sequence, a CCAAT-box in the adenovirus late promoter is necessary for termination of the upstream gene (Connelly and Manley 1989).…”

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

“…CPSF can bind to TFIID and to the body of pol II suggesting the hypothesis that CPSF binds first to the promoter via TFIID and is then transferred to pol II 30 , 31. During elongation, CPSF is proposed to associate with pol II in a manner that excludes CstF and later, CstF is recruited, at a pause site following transcription of the AAUAAA element 30,32 . Pausing has been detected downstream of the β-actin gene where it has been linked to transcription termination 33 .…”

Poly(A) signal-dependent degradation of unprocessed nascent transcripts accompanies poly(A) signal-dependent transcriptional pausing in vitro

“…The first discernable consequence of transcription across the mammalian poly(A) signal is a reduction in the rate of elongation by the polymerase due to the interaction of CPSF with the poly(A) signal hexamer (Orozco et al 2002;Park et al 2004;Nag et al 2006Nag et al , 2007. This poly(A) signaldependent pausing can be seen in the 39 flanks of genes across the genome (Gromak et al 2006;Boireau et al 2007;Glover-Cutter et al 2008;Lian et al 2008) and has been proposed to coincide with checkpoint activity that leads either to cleavage at the poly(A) site or, alternatively, to continued transcription, or to degradation of the transcript (Orozco et al 2002;Rigo et al 2005;Nag et al 2007).…”

“…For example, the relatively weak SV40 early poly(A) signal supports mRNA production less than one-fifth as efficiently in mammalian cells as does the stronger SV40 late poly(A) signal, even when both are transcribed at the same rate (Carswell and Alwine 1989). Yet, although most transcripts carrying SV40 early poly(A) sites do not get processed at those sites, little or no RNA carrying uncleaved poly(A) sites can be found in the steadystate nuclear RNA from such cells (Connelly and Manley 1988;Chao et al 1999;Park et al 2004;Nag et al 2006). Therefore, this unprocessed RNA is exceedingly unstable.…”