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Regulated Intron Retention and Nuclear Pre-mRNA Decay Contribute to PABPN1 Autoregulation - PubMed

Regulated Intron Retention and Nuclear Pre-mRNA Decay Contribute to PABPN1 Autoregulation

Danny Bergeron et al. Mol Cell Biol. 2015 Jul.

Abstract

The poly(A)-binding protein nuclear 1 is encoded by the PABPN1 gene, whose mutations result in oculopharyngeal muscular dystrophy, a late-onset disorder for which the molecular basis remains unknown. Despite recent studies investigating the functional roles of PABPN1, little is known about its regulation. Here, we show that PABPN1 negatively controls its own expression to maintain homeostatic levels in human cells. Transcription from the PABPN1 gene results in the accumulation of two major isoforms: an unspliced nuclear transcript that retains the 3'-terminal intron and a fully spliced cytoplasmic mRNA. Increased dosage of PABPN1 protein causes a significant decrease in the spliced/unspliced ratio, reducing the levels of endogenous PABPN1 protein. We also show that PABPN1 autoregulation requires inefficient splicing of its 3'-terminal intron. Our data suggest that autoregulation occurs via the binding of PABPN1 to an adenosine (A)-rich region in its 3' untranslated region, which promotes retention of the 3'-terminal intron and clearance of intron-retained pre-mRNAs by the nuclear exosome. Our findings unveil a mechanism of regulated intron retention coupled to nuclear pre-mRNA decay that functions in the homeostatic control of PABPN1 expression.

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Figures

FIG 1
FIG 1

Increased gene dosage of PABPN1 results in reduced levels of endogenous PABPN1 protein and mRNA. (A) Western blot analysis using extracts from untreated (−) or doxycycline-treated (+) transgenic (Tg) cell lines that express GFP-PABPN1 (lanes 1 and 2) and GFP-PRMT3 (lanes 3 and 4). (B) Quantification of endogenous PABPN1 protein in the presence and absence of doxycycline in the indicated cell lines. ****, P < 0.0001 by Student's t test. (C) Northern blot analysis using RNA probes complementary to PABPN1 exon 2 (lanes 1 and 2) and intron 6 (lanes 3 and 4) sequences, using total RNA from HEK293T cells treated with PABPN1-specific (lanes 2 and 4) and control (lanes 1 and 3) siRNAs. (D) RNA-seq read distribution along the PABPN1 gene from human HeLa cells. The bottom row shows the RefSeq gene annotation for the corresponding exons (E1 to E7) and introns (I1 to I6). (E) Schematic of primers used to measure the levels of spliced and unspliced PABPN1 transcripts by RT-qPCR. (F and G) RT-qPCR analysis measuring the relative fraction of total PABPN1 transcript that is spliced mRNA (F) and unspliced pre-mRNA (G) using RNA from the indicated induced (+Doxy) and noninduced (−Doxy) transgenic cell lines. Data are expressed relative to those for the uninduced control (−Doxy). **, P < 0.01 by Student's t test.

FIG 2
FIG 2

3′-Terminal intron of PABPN1 is required for autoregulation. (A) Schematic of the GFP-6/7 minigene construct in which 144 bp of exon 6, intron 6, and exon 7 was fused to the GFP coding sequence. Exon 7 consists of 37 bp of coding sequences, a TAA stop codon, and ∼900 bp of 3′ untranslated region. (B) Western blot analysis using extracts from untreated (−) and doxycycline-treated (+) GFP-PABPN1 (lanes 1 to 4) and GFP-PRMT3 (lanes 5 to 8) transgenic (Tg) cell lines that previously were transfected with the GFP-6/7 (lanes 3 and 4 as well as 7 and 8) or GFP (lanes 1 and 2 as well as 5 and 6) constructs. (C) GFP and GFP-6/7 protein levels were normalized to tubulin and expressed relative to those for the uninduced control (−Doxy). ***, P < 0.001 by Student's t test. (D) Northern blot analysis of the indicated transcripts using total RNA prepared from untreated (−) and doxycycline-treated (+) GFP-PABPN1 (lanes 1 to 6) and GFP-PRMT3 (lanes 7 and 8) transgenic cell lines that previously were transfected with the GFP-6/7 (lanes 5 to 8) or GFP (lanes 3 and 4) constructs, including a mock transfection control (lanes 1 and 2). (E) Western blot of the indicated proteins using extracts from untreated (−) and doxycycline-treated (+) GFP-PABPN1 transgenic cells that previously were transfected with wild-type (lanes 3 and 4) and intronless (lanes 5 and 6) GFP-6/7 constructs. Cells also were cotransfected with a vector expressing GFP to control for transfection efficiency. (F) GFP-6/7 protein levels were normalized to those for tubulin and GFP (to control for transfection efficiency) and expressed relative to those for the uninduced control (−Doxy). **, P < 0.01 by Student's t test. (G) Northern blot analysis of the indicated transcripts using total RNA prepared from untreated (−) and doxycycline-treated (+) GFP-PABPN1 Tg cells that previously were transfected with wild-type (lanes 1 and 2) and intronless (lanes 3 and 4) GFP-6/7 constructs. (H) Quantification of spliced GFP-6/7 transcript normalized to RPS2 mRNA and expressed relative to levels for the uninduced control (−Doxy). **, P < 0.01 by Student's t test.

FIG 3
FIG 3

Inefficient splicing of the PAPBN1 terminal intron is necessary for PABPN1 autoregulation. (A) The 5′ ss sequences of wild-type (WT) and mutated (5′ MUT) PABPN1 intron 6 are shown. Nucleotide changes introduced in intron 6 sequence to enhance splicing efficiency are shown in green. Nucleotide positions are numbered and indicated under each nucleotide. ΔG, predicted free energy of 5′ ss. (B) Northern blot analysis using total RNA prepared from untreated (−) and doxycycline-treated (+) GFP-PABPN1 transgenic (Tg) cells that previously were transfected with wild-type (lanes 1 and 2) and 5′ ss mutant (lanes 3 and 4) GFP-6/7 constructs. (C) Quantification of GFP-6/7 unspliced/spliced ratios from Northern blot data and expressed relative to those for the uninduced control (−Doxy). *, P < 0.05 by Student's t test. (D) Western blot analysis of the indicated proteins using extracts from untreated (−) and doxycycline-treated (+) GFP-PABPN1 transgenic cells that previously were transfected with wild-type (lanes 1 and 2) and 5′ ss mutant (lanes 3 and 4) GFP-6/7 constructs. (E) GFP-6/7 protein levels were normalized to those for tubulin and GFP (to control for transfection efficiency) and expressed relative to those for the uninduced control (−Doxy). *, P < 0.05 by Student's t tests. (F) Schematic of GFP-6/7 constructs with PABPN1, RPS2, and HNRNPK 3′-terminal introns used to study PABPN1-dependent regulation. (G and H) Northern blot (G) and Western blot (H) analyses of extracts prepared from untreated (−) and doxycycline-treated (+) GFP-PABPN1 transgenic cells that previously were transfected with GFP-6/7 constructs containing the PABPN1 (lanes 1 and 2), RPS2 (lanes 3 and 4), and HNRNPK (lanes 5 and 6) introns. Cells also were cotransfected with a vector expressing GFP to control for transfection efficiency.

FIG 4
FIG 4

PABPN1 autoregulation does not require canonical 3′-end processing and polyadenylation. (A) Schematic diagram of the GFP-6/7 ribozyme construct. (B) Northern blot analysis of total (lanes 1 and 3) and oligo(dT)-selected (lanes 2 and 4) RNA prepared from cells transfected with DNA constructs that express wild-type (lanes 1 and 2) and ribozyme-processed (lanes 3 and 4) GFP-6/7 transcript. (C) Northern blot analysis of total RNA prepared from untreated (−) and doxycycline-treated (+) GFP-PABPN1 transgenic (Tg) cells that previously were transfected with wild-type (lanes 1 and 2) and ribozyme-processed (lanes 3 and 4) GFP-6/7 constructs. (D) Quantitative RT-PCR analysis of spliced GFP-6/7 mRNA produced from wild-type and ribozyme constructs. GFP-6/7 mRNA levels were normalized to RPS2 mRNA and expressed relative to those for the uninduced control (−Doxy). ***, P < 0.001 by Student's t test. (E) Western blot analysis using extracts from untreated (−) and doxycycline-treated (+) GFP-PABPN1 transgenic cells that previously were transfected with wild-type (lanes 1 and 2) and ribozyme (lanes 3 and 4) GFP-6/7 constructs. (F) GFP-6/7 protein levels were normalized to those for tubulin and expressed relative to those for the uninduced control (−Doxy). ***, P < 0.001 by Student's t test.

FIG 5
FIG 5

Adenosine-rich region in the 3′ UTR of the PABPN1 transcript is important for PABPN1 autoregulation. (A) A 50-nt, adenosine-rich region from the human PABPN1 mRNA is shown. C-to-A changes introduced in adenosine repeats are shown in red and are referred to as A-MUT1 and A-MUT2. (B) Increasing amounts of GST (lanes 2 to 5) and GST-PABPN1 (lanes 6 to 9) were incubated with a 67-nt RNA probe that includes the A repeats of the PABPN1 3′ UTR. The position of free probe and PABPN1-bound complexes is shown on the left. (C) Equal amounts of GST-PABPN1 were incubated with the A-rich 3′ UTR probe in the presence of increasing amounts of cold wild-type (WT, lanes 3 and 4) and C-to-A mutated (A-MUT1, lanes 5 and 6; A-MUT2, lanes 7 and 8) 3′ UTR probes or without any competing nucleic acid (lane 2). (D) Western blot analysis using extracts from untreated (−) and doxycycline-treated (+) GFP-PABPN1 transgenic (Tg) cells that previously were transfected with wild-type (lanes 3 and 4) and C-to-A mutated (lanes 5 to 8) GFP-6/7 constructs. (E) GFP-6/7 protein levels were normalized to those of tubulin and GFP (to control for transfection efficiency) and expressed relative to those for the uninduced control (−Doxy). P < 0.01 (**) and P < 0.05 (*) by Student's t tests. (F) Northern blot analysis using total RNA prepared from untreated (−) and doxycycline-treated (+) GFP-PABPN1 transgenic cells that previously were transfected with wild-type (lanes 5 and 6) and C-to-A mutated (lanes 7 to 10) GFP-6/7 constructs. (G) RT-PCR analysis of unspliced/spliced ratios for wild-type and C-to-A mutant constructs expressed relative to those for the uninduced control (−Doxy). **, P < 0.01 by Student's t test. (H) RT-PCR analysis of unspliced/spliced ratios for wild-type and C-to-A mutant constructs expressed under uninduced conditions (−Doxy). P < 0.01 (**) and P < 0.05 (*) by Student's t tests.

FIG 6
FIG 6

SRSF10, hnRNP H, and hnRNP A2/B1 modulate PABPN1 intron 6 splicing. (A) Western blot analysis using total extracts prepared from HEK293T cells that were transfected with constructs expressing GFP (lane 1), GFP-SRSF10 (lane 2), and GFP-hnRNP C (lane 3). (B) RT-qPCR analysis of the endogenous PABPN1 unspliced/spliced ratio using total RNA prepared from HEK293T cells that previously were transfected with constructs expressing GFP, GFP-SRSF10, and GFP-hnRNP C. ***, P < 0.001 by Student's t test. (C and D) RIP assays were performed using GFP and GFP-SRSF10. Pre-mRNA association (IP/input ratio) for GFP and GFP-SRSF10 was analyzed by RT-qPCR, and the values then were set to 1.0 for the control GFP purification. (E) RT-qPCR analysis of RIP assays using GFP and GFP-SRSF10 that were expressed in HEK293T cells that conditionally express Flag-PABPN1 using a doxycycline (Doxy)-sensitive promoter. PABPN1 pre-mRNA association was measured as described for panel D, and the values were sent to 1.0 for the GFP control that was purified from uninduced cells (−Doxy). The P value was calculated using Student's t test. (F) Western blot validation of a RIP assay, as described for panel E, for GFP immunoprecipitations (IP; lanes 5 to 8) and whole-cell extracts (input; lanes 1 to 4). (G) Western blot validation of RNAi-mediated depletion of hnRNP A2/B1 (lane 2), hnRNP H (lane 4), and hnRNP C (lane 6). (H) RT-qPCR analysis of PABPN1 unspliced/spliced ratios using total RNA prepared from HEK293T cells treated with siRNAs specific to hnRNP H, hnRNP A2/B1, and hnRNP C, as well as a nontarget control siRNA. P < 0.001 (***), P < 0.01 (**), and P < 0.05 (*) by Student's t test.

FIG 7
FIG 7

PABPN1 autoregulation requires the nuclear RNA exosome. (A) Western blot validations of RNAi-mediated depletion of UPF1 (lane 2), hRRP40 (lane 4), hMTR4 (lane 6), and XRN2 (lane 8). (B and C) Quantitative RT-PCR analysis of spliced PABPN1 mRNA (B) and HPRT mRNA (C) levels using total RNA prepared from HEK293T cells treated with siRNAs specific to hRRP40, hMTR4, UPF1, and XRN2, as well as a nontarget (NT) control siRNA. To specifically assay for PABPN1-dependent effects, data were normalized to those for RPS2 mRNA and expressed relative to those for the uninduced conditions (−Doxy). P < 0.01 (**) and P < 0.05 (*) by Student's t tests.

FIG 8
FIG 8

Model for PABPN1 autoregulation. (A) The homeostatic control of PABPN1 expression involves competition between pre-mRNA decay by the exosome and splicing of the 3′-terminal intron by the spliceosome. Splicing regulators and PABPN1 influence the equilibrium between pre-mRNA splicing and decay. (B) When the nuclear concentration of PABPN1 is high, binding of PABPN1 to an adenosine (A)-rich region in its 3′ UTR interferes with splicing of the 3′-terminal intron, which promotes pre-mRNA decay by the exosome.

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