Poly(A) binding protein nuclear 1 levels affect alternative polyadenylation

Abstract

The choice for a polyadenylation site determines the length of the 3'-untranslated region (3'-UTRs) of an mRNA. Inclusion or exclusion of regulatory sequences in the 3'-UTR may ultimately affect gene expression levels. Poly(A) binding protein nuclear 1 (PABPN1) is involved in polyadenylation of pre-mRNAs. An alanine repeat expansion in PABPN1 (exp-PABPN1) causes oculopharyngeal muscular dystrophy (OPMD). We hypothesized that previously observed disturbed gene expression patterns in OPMD muscles may have been the result of an effect of PABPN1 on alternative polyadenylation, influencing mRNA stability, localization and translation. A single molecule polyadenylation site sequencing method was developed to explore polyadenylation site usage on a genome-wide level in mice overexpressing exp-PABPN1. We identified 2012 transcripts with altered polyadenylation site usage. In the far majority, more proximal alternative polyadenylation sites were used, resulting in shorter 3'-UTRs. 3'-UTR shortening was generally associated with increased expression. Similar changes in polyadenylation site usage were observed after knockdown or overexpression of expanded but not wild-type PABPN1 in cultured myogenic cells. Our data indicate that PABPN1 is important for polyadenylation site selection and that reduced availability of functional PABPN1 in OPMD muscles results in use of alternative polyadenylation sites, leading to large-scale deregulation of gene expression.

Figure 1.

Single molecule polyadenylation site sequencing method. The procedure of the method is detailed in the results and materials and methods sections. Red and grey boxes represent magnetic stands used to capture the oligo(dT) magnetic beads. Green anchors represent biotin labels, and red anchors represent fluorescent labels for nucleotide analogs.

Figure 2.

Alternative polyadenylation analysis in mouse muscles. (A) Width of polyadenylation site clusters. The x-axis represents the width of the cluster on a logarithmic scale, the y-axis represents the number of polyadenylation site-clusters. (B) Bar graph showing the number of polyadenylation sites detected per transcript. Only polyadenylation sites mapping to the expanded 3′-UTR, and covered by at least two reads are shown. Pie chart shows the percentage of transcripts containing at least two polyadenylation sites.

Figure 3.

Altered polyadenylation site usage in A17.1 mice. (A) A screenshot of USCS Genome Browser displaying polyadenylation sites in Psdm14 gene. The y-axis represents the coverage of the peaks, corresponding to the number of reads mapping at each polyadenylation site. A control (FVB) and an A17.1 mouse are shown in independent traces. Below the coverage tracks, the four annotated transcripts are shown. The longest transcript (ENSMUST00000028278) contains a 3′-UTR of 297 nucleotides. There are two major polyadenylation sites in this region (indicated by arrows), a distal one (peak location chr2:61,638,431-61,638,432) at the annotated 3′-end of the transcript and a proximal one located 276 nucleotides upstream and just 23 nucleotides downstream of the stop codon (peak location chr2:61,638,155-61,638,156). (B) Volcano plots showing transcripts variants containing the distal polyadenylation site (left panel) or more proximal polyadenylation sites (right panel). The y-axis represents the −¹⁰log of the multiple testing adjusted P-values, while the x-axis represents the ratio of expression in A17.1 over wild-type mice on a logarithmic scale (base 2). The blue line represents a P-value threshold of 0.05. The outliers present in the graphs represent data points were the total counts in one of the two groups is zero. (C) Pabpn1 expression level in quadriceps muscles of A17.1 mice and FVB mice, as measured by qRT-PCR with primers measuring both endogenous and exogenous Pabpn1. (D) The ratio of proximal PCR over distal PCR products in A17.1 mice (black bars) and FVB mice (white bars), as measured by qRT–PCR. Values are means ± standard deviation for n = 6 mice per group (*P < 0.05, **P < 0.01).

Figure 4.

Switches in polyadenylation site induce changes in gene expression. (A) Overlap between deregulated transcripts and transcripts with changes in polyadenylation site usage. (B) The overlap is shown for downregulated and upregulated transcripts separately. Indicated P-values were calculated with Fisher’s test.

Figure 5.

Modulation of PABPN1 expression levels induces changes in polyadenylation site usage in C2C12 cells. (A) Total (endogenous and exogenous) Pabpn1 mRNA levels in C2C12 myotubes transduced with CMV-GFP (white) and YFP-Ala16-PABPN1 (black). (B) Ratio of proximal:distal PCR products, representing a combination of short and long 3′-UTRs respectively, in CMV-GFP (white) and YFP-Ala16-PABPN1 (black) transduced myotubes. (C) Total Pabpn1 mRNA levels in C2C12 myotubes transduced with CMV-GFP (white), YFP-Ala16-PABPN1 (black) and CFP-Ala10-PABPN1 (grey). (D) Relative proximal:distal ratio between YFP-Ala16-PABPN1:CMV-GFP (black) and CFP-Ala10-PABPN1: CMV-GFP (grey), compared with the control CMV-GFP (white). (E) Pabpn1 mRNA levels in C2C12 myoblasts transduced with TRCN0000102536 (grey) and TRCN0000000121 (black) shRNAs targeting Pabpn1 and cells transduced with the control shRNA H1-Ctl (white). (F) Proximal:distal ratio for C2C12 myoblasts transduced with the two different sh-RNAs against Pabpn1 (grey, black) and the control shRNA H1-Ctl (white). Values are means ± standard deviation for 3 different wells. All experiments were repeated multiple times with similar results.

Figure 6.

Transcripts with distal polyadenylation site are enriched in PABPN1 immunoprecipitated RNA. (A) The proximal:distal ratio for input RNA (white) and PABPN1-immunoprecipitated RNA (black) was determined by qRT–PCR using the same primer sets, as in Figure 5. Values are means ± standard deviation for three independent experiments. (B) Western blot analysis shows levels of PABPN1 and tubulin, loading control, in the input. Levels of immunoprecipitated (IP) PABPN1 in C2C12 and tubulin, as negative control for immunoprecipitation, are shown. An IP without anti-PABPN1 antibody (−) was used as negative control.