Poly(A) signal-dependent degradation of unprocessed nascent transcripts accompanies poly(A) signal-dependent transcriptional pausing in vitro.
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 3'-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.
Kazerouninia
Amir
A
Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095-1569, USA.
Ngo
Benson
B
Martinson
Harold G
HG
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D011061:Poly A / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D020871:RNA Stability / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology*; D035602:Transcriptional Elongation Factors / Q000378:metabolism / Q000502:physiology
D012322:RNA Precursors; D035602:Transcriptional Elongation Factors; D011061:Poly A; D012321:DNA-Directed RNA Polymerases