RNA polymerase II kinetics inpolopolyadenylation signal selection

Abstract

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3 0 untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3 0 UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation.

Ending the message: poly(A) signals then and now

“…In the case of the gene Polo, with its two well-defined PAS, a weak proximal and stronger distal PAS are regulated such that the distal PAS is required for higher levels of Polo gene expression. Interestingly, flies expressing a slow Pol II mutant show a clear shift to usage of the proximal Polo PAS as well as several other tested examples of fly APA (Pinto et al 2011).…”

Integrating transcription kinetics with alternative polyadenylation and cell cycle control

“…We constructed transgenic flies in a polo 9 background deleting either pA1 or pA2 and performed a developmental analysis of the transgenic individuals. Surprisingly, we found that deletion of the distal pA signal had a profound biological impact: transgenic flies without polo pA2 (DpA2) died at the pupa stage of development with severe abdominal abnormalities, 19 revealing yet another level of polo regulation, through alternative polyadenylation.…”

“…In a recently published study addressing genes containing two pA signals located in tandem in the 3'-untranslated region, we showed that a "slow" Pol II preferentially uses the most proximal pA signal in several endogenous Drosophila genes. 19 Using chromatin immunoprecipitation (ChIP), reverse transcription coupled with real-time polymerase chain reaction (RT-qPCR) and 3' rapid amplification of cDNA ends (RACE) experiments, we showed that the decrease in the Pol II elongation rate in the "slow" Pol II mutant RpII215, when compared with wild-type flies, caused a pA switch in six out of eight genes tested. The longer period that the "slow" Pol II needs for transcription might allow for a longer exposure of the first pA signal to cleavage/polyadenylation factors thus enhancing polyadenylation before the enzyme reaches the second pA signal.…”

Alternative polyadenylation: New insights from global analyses

“…Indeed, it has recently been shown that APA is modulated by the transcription elongation rate. Using the Drosophila polo mRNAs as a model, Pinto et al (2011) showed that a RNAP II mutant with slower elongation rate kinetically favors the recognition of the proximal PAS. In contrast, in cells expressing the wild-type RNAP II, the intrinsic advantage of the proximal PAS becomes less significant, and as a result, the relative usage of the distal PAS increases (Pinto et al 2011).…”