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. 2011 May 20;30(12):2431-44.
doi: 10.1038/emboj.2011.156.

RNA polymerase II kinetics in polo polyadenylation signal selection

Pedro A B Pinto  1 , Telmo Henriques, Marta O Freitas, Torcato Martins, Rita G Domingues, Paulina S Wyrzykowska, Paula A Coelho, Alexandre M Carmo, Claudio E Sunkel, Nicholas J Proudfoot, Alexandra Moreira
Affiliations

RNA polymerase II kinetics in polo polyadenylation signal selection

Pedro A B Pinto et al. EMBO J. .

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' untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3'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.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
RNA Pol II kinetics with a slower transcription elongation rate affects pA site selection. (A) Pol II ChIP across polo and polo-snap intergenic region. In the top panel, a schematic diagram is shown where polo and snap genes are depicted to scale (adapted from genome.ucsc.edu). ChIP was performed using α-Rpb3 antibody on wild-type (w1118) and slow Pol II (RpII215 mutant, C4 mutation) adult flies chromatin. Numbers below each graph bar represent the position of real-time PCR primers. (B) RpII215 mutant generates similar levels of polo mRNA as compared with w1118. Graph represents RT–qPCR quantification of polo mRNAs in adult flies, relative to rp49 mRNA. (C) RpII215 mutant presents similar levels of Polo protein as w1118. Western blot from total protein extracts of w1118 and RpII215 third instar larvae brains. MA294 Polo and DM1A α-tubulin antibodies were used. Quantification was made by densitometry (see Materials and methods) and Polo/α-tubulin ratio was set at 1 for w1118. (D) Representative gel of fractionated 3′RACE products of polo mRNA from w1118 and RpII215 adult flies is shown. Arrows indicate the bands corresponding to polo pA1 and pA2; −RT is a control reaction without reverse transcriptase. In both panels, phased-anchored oligodT was used for reverse transcription, as depicted in the diagram. (E) RpII215 shows an increase in the ratio of total/pA2 mRNAs. Diagram shows primer positions for qPCR analysis. Levels of total polo mRNAs and pA2 mRNAs were measured by real-time PCR and their ratio (total/pA2) in adult flies is shown. (F) RpII215 shows an increase in proximal site usage in several genes. RT–qPCR quantification was performed as in (E). cDNA synthesis was performed with random primers (see Materials and methods). The ratio for w1118 was set at 1. For all the panels, error bars show s.e.m. from at least three independent experiments.
Figure 2
Figure 2
Deletion of polo distal pA2 signal causes abnormal formation of the fly abdomen. (A) Schematic representation of the gfp-polo, ΔpA1 and ΔpA2 transgenes driven by the endogenous polo promoter. The two pA signals ATTAAA and ATATAA are indicated. In the ΔpA1 transgene, the proximal pA signal was mutated to GTTAAC. (B) ΔpA1 and ΔpA2 transgenes utilize pA2 and pA1 signals, respectively, as indicated by the arrows. Northern blot of w1118, gfp-polo, ΔpA2 (lines 2.3 and 5) and ΔpA1 (lines 12.1 and 9.1) from third instar larvae brains total RNA. The asterisks indicate the two polo mRNAs present in w1118, the black arrows the shorter transcripts present in ΔpA2 lines and the open arrows the longer transcripts present in ΔpA1 lines. All fly lines analysed contain the endogenous polo mRNAs that serve as internal controls (denoted by *). (C) polo 3′UTR sequence. Bold sequence indicates proximal (pA1) and distal (pA2) pA signals. The arrows indicate the cleavage site of the transcripts produced by usage of pA1 and pA2. (D) polo pA2 is required for abdominal development. Dorsal view of female abdomens from w1118 control (panel 1); the seven tergites of the adult abdomen are indicated as A1–A7. Panels 2–4: gfp-polo, ΔpA1 (line 12.1) and ΔpA2 (line 2.3) individuals in a polo9/TM6B background. Panels 5–7: gfp-polo, ΔpA1 (line 12.1) and ΔpA2 (line 2.3) individuals in a polo9 background. (E) polo mRNA quantification by RT–qPCR in the transgenic fly lines in a polo9 background. Total RNA was extracted from two adult flies of each genotype (for ΔpA2;polo9, two escapers were used) in a polo9 background, and from w1118 flies. After cDNA synthesis with random hexamers, RNA levels were quantified using gfp-specific primers for amplification of the transgene-derived transcripts (light grey bars) or primers for exons 4 and 5 for amplification of the total polo RNAs present (dark grey bars). In all genotypes, rp49 was used for normalization. The difference between total polo (dark grey bars) and gfp-polo (light grey bars) corresponds to polo9 contribution. Error bars show s.e.m. from at least three independent experiments.
Figure 3
Figure 3
ΔpA2 transgene causes a failure in abdominal histoblasts proliferation and produces lower Polo protein levels in histoblasts. (A) Pupa epidermis from gfp-polo, ΔpA1 or ΔpA2 pupae transgenes in a polo9 homozygous background was dissected at 26–27 h after pupa formation and stained with DAPI. gfp-polo;polo9 pupa epidermis exhibited large polyploid nuclei (circled), the larva epidermis cells (LECs). LECs were surrounded by smaller cells (highlighted by dotted lines), which are the proliferating abdominal histoblasts. ΔpA1;polo9 pupa epidermis show a pattern very similar to that observed in gfp-polo;polo9 tissues. In ΔpA2;polo9, only LECs are observed, indicating that at this stage, pupa epidermis is mainly composed of larval cells. Scale bar is 50 μm. (B) GFP–Polo expression (green) in abdominal histoblasts in pupae epidermis (26–27 h after pupa formation). Pupae from gfp-polo, ΔpA1 (line 12.1) and ΔpA2 (line 2.3) transgenic flies in a polo9/TM6B background were collected and the epidermis was dissected. The DNA (red) was stained with DAPI. The yellow dotted surrounding area encompasses some of the histoblast cells present in each image. Scale bar is 10 μm. (C) The levels of GFP–Polo were determined in the abdomen histoblasts, by quantification of the GFP fluorescence levels in gfp-polo, ΔpA1 (line 12.1) and ΔpA2 (line 2.3) pupae in a polo9/TM6B background, 26–27 h after pupa formation. (D) The levels of gfp-polo transcripts were determined by RT–qPCR in gfp-polo, ΔpA1 (line 12.1) and ΔpA2 (line 2.3) whole pupae in a polo9/TM6B background, 26–27 h after pupa formation. The levels of gfp-polo RNAs were normalized to rp49 RNA. Error bars represent s.e.m. from three independent experiments.
Figure 4
Figure 4
polo pA2 RNA is stable and the corresponding 3′UTR confers higher translability to a reporter gene in Drosophila cells. (A) Endogenous polo pA2 RNA is stable. The decay of the endogenous polo RNAs and Vha68-1 RNA was monitored at the indicated time points after addition of actinomycin D (5 μg/ml). The levels of polo RNAs were normalized to rp49 RNA in three independent experiments and are plotted against time. RNA half-lives (t1/2), calculated from the decay curves, are longer than 6 h, except for Vha68-1 that is ∼30 min. (B) Representation of the firefly luciferase reporter constructs with the shorter polo 3′UTR (pA1) or the longer polo 3′UTR (pA2) (pA1 signal mutation: ATTAAA to GTTAAC). The region for specific qPCR primers is underlined. (C) pA1 produces three-fold less protein than pA2. Kc cells were transfected with the pA1 and pA2 reporters and 3 days later firefly luciferase activity and the corresponding mRNA levels were measured. Y axis represents luciferase values normalized to the mRNA levels. Error bars represent s.e.m. from at least three independent experiments.
Figure 5
Figure 5
Overexpression of polo leads to an increase in pA1 utilization and a reduction in GFP–Polo protein levels. (A) MZ1061-Gal4;UAS-polo:polo9 overexpresses Polo. L3 brains were analysed by western blot and a 2.3-fold increase in Polo was observed in comparison with the control—w1118. Quantification was made by densitometry (see Materials and methods) and Polo/α-tubulin ratio was set at 1. Polo was detected with MA294 and α-tubulin with DM1A antibodies. (B) Overexpression of Polo causes a switch in the pAs ratio. Total RNA was extracted from L3 larvae brains of the MZ1061-Gal4;gfp-polo:polo9/UAS-polo:polo9 progeny (labelled as overexpression in the figure). The strain MZ1061-Gal4;gfp-polo:polo9 was used as control. Total/pA2 mRNA levels were quantified by RT–qPCR with specific primers for both transcripts in the coding sequence and primers between the two pAs. A 2.5-fold change was detected in comparison with the control. (C) GFP–Polo protein expression was analysed by western blot, using the same conditions as in (A). When polo is overexpressed, a four-fold decrease in GFP–Polo is observed.
Figure 6
Figure 6
Proposed working model for the role of Pol II kinetics on pA site selection. (A) When Pol II elongation rate is low, Pol II spends more time transcribing polo allowing the pA factors (represented by a triangle and an hexagon) to assemble on the pA1 signal presented by the nascent pre-mRNA, leading to an increase in pA1 utilization, and therefore producing four-fold more polo pA1 mRNA. In conditions where Pol II processivity is high, Pol II transcribes more rapidly through pA1 and pA2. Under these conditions, both pA1 and pA2 signals are present on the nascent transcript and exposed to the polyadenylation machinery at the same time. Consequently, both polo pA1 and pA2 mRNAs are formed. (B) The longer polo mRNA, produced by pA2 signal selection and containing a longer 3′UTR, is more efficiently translated into protein than the shorter pA1 mRNA. Consequently, selection of pA2 is important for the viability of the fly. Levels of Polo protein are controlled by a negative feedback loop mechanism via pA site selection; in the presence of high levels of Polo, pA1 signal is predominantly selected, causing a decrease in the levels of Polo protein produced.
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