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

Abstract

The poly(A) signal has long been known for its role in directing the cleavage and polyadenylation of eukaryotic mRNA. In recent years its additional coordinating role in multiple related aspects of gene expression has also become increasingly clear. Here we use HeLa nuclear extracts to study two of these activities, poly(A) signal-dependent transcriptional pausing, which was originally proposed as a surveillance checkpoint, and poly(A) signal-dependent degradation (PDD) of unprocessed transcripts from weak poly(A) signals. We confirm directly, by measuring the length of RNA within isolated transcription elongation complexes, that a newly transcribed poly(A) signal reduces the rate of elongation by RNA polymerase II and causes the accumulation of elongation complexes downstream from the poly(A) signal. We then show that if the RNA in these elongation complexes contains a functional but unprocessed poly(A) signal, degradation of the transcripts ensues. The degradation depends on the unprocessed poly(A) signal being functional, and does not occur if a mutant poly(A) signal is used. We suggest that during normal 39-end processing the uncleaved poly(A) signal continuously samples competing reaction pathways for processing and for degradation, and that in the case of weak poly(A) signals, where poly(A) site cleavage is slow, the default pathway to degradation predominates.

RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3′ UTRs

“…Defects in 39 processing in yeast are known to be coupled to exosome-mediated degradation of the unprocessed RNAs (Hilleren et al 2001;Milligan et al 2005;Kazerouninia et al 2010). We therefore suspected that the observed decreased accumulation of ARE-containing transcripts could be a consequence of exosomal degradation of the uncleaved transcripts.…”

Poly(A) Signal-Dependent Transcription Termination Occurs through a Conformational Change Mechanism that Does Not Require Cleavage at the Poly(A) Site

“…To develop a fundamental understanding of eukaryotic PADT, we have focused for a number of years on the core PAS and basal PADT. Basal PADT requires only a single element-the PAS (Orozco et al, 2002), which alone can direct pausing and then termination of transcription (Orozco et al, 2002;Nag et al, 2007;Kazerouninia et al, 2010) with polymerases dissociating from the template according to apparent first-order kinetics downstream of the PAS (Orozco et al, 2002;Kim and Martinson, 2003).…”

Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints

“…These control pathways "enforce dependency in the cell cycle" by delaying progress in response to specific problems so that they can be corrected or resolved prior to division. [The term "checkpoint" is starting to be used to also describe other surveillance mechanisms not involved in cell cycle regulation including, e.g., one that prevents the expression of aberrant RNA transcripts (see references in Kazerouninia et al 2010;Eberle et al 2010)]. Cell cycle checkpoints evolved to minimize the production and propagation of genetic mistakes.…”