Loss of MBNL leads to disruption of developmentally regulated alternative polyadenylation in RNA-mediated disease

Abstract

Inhibition of muscleblind-like (MBNL) activity due to sequestration by microsatellite expansion RNAs is a major pathogenic event in the RNA-mediated disease myotonic dystrophy (DM). Although MBNL1 and MBNL2 bind to nascent transcripts to regulate alternative splicing during muscle and brain development, another major binding site for the MBNL protein family is the 3' untranslated region of target RNAs. Here, we report that depletion of Mbnl proteins in mouse embryo fibroblasts leads to misregulation of thousands of alternative polyadenylation events. HITS-CLIP and minigene reporter analyses indicate that these polyadenylation switches are a direct consequence of MBNL binding to target RNAs. Misregulated alternative polyadenylation also occurs in skeletal muscle in a mouse polyCUG model and human DM, resulting in the persistence of neonatal polyadenylation patterns. These findings reveal an additional developmental function for MBNL proteins and demonstrate that DM is characterized by misregulation of pre-mRNA processing at multiple levels.

Figure 1. MEF PolyA Site Shifts Following Mbnl Depletion

(A) Mbnl proteins are localized primarily in the nucleus in MEFs. Immunoblot analysis of nuclear (Nuc) and cytoplasmic (Cyt) subcellular fractions demonstrated that Mbnl proteins were predominantly nuclear. Elavl1/HuR was included as a nuclear RBP control and LDHA as a cytoplasmic (Cyt) marker. (B) Immunoblot validation of MEF Mbnl knockouts and knockdowns. Mbnl1-3 protein levels were analyzed by immunoblotting of whole cell lysates isolated from wild type (WT), Mbnl1^ΔE3/ΔE3; Mbnl2^ΔE2/ΔE2 double KO (DKO), Mbnl1^ΔE3/ΔE3 (Mbnl1 KO), Mbnl2^ΔE2/ΔE2 (Mbnl2 KO) (left panel). Right panel shows a comparison of Mbnl3 levels in WT (longer exposure compared to left panel) and DKO MEFs following treatment with the siRNAs targeting Mbnl3 (siMbnl3). Gapdh was included as a loading control. (C) Scatter plots illustrating the shift of 3′ UTR poly(A) sites to shorter (proximal, red) versus longer (distal, blue) sites relative to the coding region in WT versus DKO (left) and WT versus DKO/3 KD MEFs (right) based on FDR <0.001 and −0.15>dI>0.15. The number of shifts (n) were: 1) DKO, proximal (n = 3466), distal (1131), total (4597), no shift (53568); 2) DKO/3KD, proximal (3626), distal (1481), total (5107), no shift (53060). (D) Wiggle plots of PolyA-seq data of two Mbnl target genes (Fosl2, Papola). The figure includes terminal exons with 3′ UTRs (thick black line) and coding regions (black box) showing pA shifts (left) and 3′ RACE gel validation (right) of WT (turquoise) and DKO (orange) MEFs. (E) Wiggle plots of Calm3 PolyA-seq (left), 3′ RACE (middle) and qRT-PCR bar graphs (right, n = 3 per sample, data represented as mean +/- SEM, **p < 0.01) for WT (turquoise), DKO (orange) and DKO/3 KD (red) MEFs.

Figure 2. Mbnl Binding Sites and Alternative Polyadenylation

(A) Pie charts showing genomic distributions of binding sites for Mbnl1, Mbnl2 and Mbnl3 in WT MEFs and Mbnl3 in Mbnl1/2 KO (Mbnl3/DKO) MEFs. (B) Venn diagram of genes that contain Mbnl binding sites (HITS-CLIP targets) showing overlap of genes that are regulated by Mbnl1-3 in MEFs. (C) Wiggle plots of HITS-CLIP (top) and PolyA-seq (bottom) highlight Mbnl binding sites that overlap and flank affected pAs in MEFs for 3′ UTRs (Calm3, Itgb1) and introns (Camk1d). For Mbnl3 HITS-CLIP, DKO MEFs were used since loss of Mbnl1 and Mbnl2 led to an increase in Mbnl3 binding to Mbnl targets. HITS-CLIP brackets indicate the number of unique tags at each site. PolyA-seq analysis was performed on WT (turquoise), DKO (orange), DKO treated with non-targeting siRNAs (DKO/NT, black) and DKO treated with Mbnl3 siRNAs (DKO/3KD, red) MEFs. (D) Mbnl polyadenylation regulatory map showing Mbnl CLIP tag density ± 200bp from the cleavage site and CLIP read density (x10³). (E) Two major functional patterns for Mbnl proteins in alternative polyadenylation.

Figure 3. Minigene Polyadenylation Reporter Analysis

(A) Minigene reporters composed of the Calm3 and Itgb1 3′UTRs (thick green lines) with the proximal (pA1) and distal (pA2) polyadenylation sites cloned downstream of the IRES-driven (grey box) luciferase coding region (turquoise box). The primer binding sites for RT-PCR and 3′ RACE (red bars), the wild type (WT) and mutant (MUT, red letters) sequences downstream (Calm3) or upstream (Itgb1) of the cleavage sites (green CA indicated for Calm3) and the AU-rich binding sites for ELAVL1/HuR (white boxes in Itgb1 3′ UTR) are also indicated. (B) Relative protein levels for MBNL1, MBNL2, MBNL3 and ELAVL1/HuR in WT MEFs compared to COSM6 cells were determined by immunoblotting. (C) Immunoblots showing myc-tagged Mbnl1 overexpression following transfection of COSM6 cells with pcDNA3.1-Mbnl1_myc. Antibodies to Mbnl1, Myc and Gapdh (loading control) are shown for cells transfected with Calm3^WT (WT) and Calm3^MUT (MUT) polyadenylation reporters without/with co-transfection with pcDNA3.1-Mbnl1_myc (+Mbnl1). (D) Mbnl proteins repress Calm3 proximal pA (pA1), and activate Itgb1 distal pA (pA2), site use. The ratio of distal to proximal pA selection (D/P) was determined by qRT-PCR for COSM6 cells transfected with the Calm3^WT (WT), Calm3^MUT (MUT), Itgb13^WT (WT), Itgb1^MUT (MUT) reporters without/with Mbnl1_myc overexpression (+Mbnl1) (data represented as mean +/- SEM, *p < 0.05, **p < 0.01). (E) Elevated Calm3 pA1 site use leads to enhanced, while loss of Itgb1 distal (pA2) results in decreased, luciferase activity. COSM6 cells were mock transfected (con) or transfected as described in (D) (data represented as mean +/- SEM, *p < 0.05, **p < 0.01). (F) RNA immunoprecipitation assay with anti-Cpsf160, anti-Cstf2 and anti-Tardbp (control) antibodies. (G) Examples (Tpm1, Slc25a4) of HITS-CLIP (top) and PolyA-Seq (bottom) showing overlap of Mbnl and Cpsf6 binding sites in wild type (WT), but not Mbnl DKO, MEFs. Enhanced Cpsf6 binding and activation of upstream pA sites were observed following loss of Mbnl activity in DKO MEFs. (H) RNA map of Cpsf6 normalized CLIP tag density (± 200 bp from the cleavage site, CS) in WT versus DKO MEFs. Polyadenylation sites without additional proximal (±300 bp) sites were selected to avoid interference.

Figure 4. APA dysregulation in the HSA^LR mouse DM1 model

(A) Mbnl1 and Mbnl2, but not Mbnl3, are expressed in WT and HSA^LR adult muscle. Immunoblots showing that Mbnl1 protein was expressed at equivalent levels in both WT and HSA^LR quadriceps while Mbnl2 expression was upregulated in HSA^LR compared to WT adult and Mbnl3 was not expressed at a detectable level. (B) Mbnl1 and Mbnl2 proteins colocalize with CUG^exp RNAs in HSA^LR muscle. Immunofluorescence (green), using anti-Mbnl1 (α-Mbnl1) and anti-Mbnl2 (α-Mbnl2) antibodies, and RNA-FISH (red), using Cy3-conjugated (CAG)₁₀ to detect CUG^exp RNA, of quadriceps muscle sections (nuclear DNA is stained with DAPI, blue). The Mbnl2 nuclear/cytoplasmic ratio was unchanged in WT versus HSA^LR so the fluorescence signal in HSA^LR (α-Mbnl2) versus no signal in WT reflects increased Mbnl2 concentration in HSA^LR RNA foci. (C) Venn diagram of overlapping pA changes in WT neonatal (WT P1) versus WT adult (light blue) and WT adult versus HSA^LR adult muscle (green) and the overlap (dark blue). (D) Scatter plots illustrating 3′ UTR shortening (proximal, red) and lengthening (distal, blue) in WT neonatal (P1) and HSA^LR quadriceps muscles versus WT adult based on FDR < 0.05 and −0.15>dI>0.15. The number of shifts (n) were: 1) P1, proximal (2588), distal (1360), total (3948), no shift (42601); 2) HSA^LR, proximal (n = 641), distal (342), total (983); no shift (45566). (E) Concordance of APA patterns between WT neonatal and HSA^LR adult muscle. The distal to proximal pA site (D/P) ratios were determined for Fxr1, Ndrg3, Pdlim5, Sgcg and Tuba4a by PCR for WT adult (blue), WT neonates (P1, green) and HSA^LR adult (red) muscles within the top 10 genes having pA shifts and Mbnl1 CLIP tags (data represented as mean +/- SEM, **p < 0.01, ***p < 0.001). The majority of Mbnl RNA targets, except Sgcg and Pdlim5, show significant concordant shifts between WT P1 and HSA^LR.

Figure 5. Reversion to Fetal APA Patterns in HSA^LR Mice

(A) Pie chart of Mbnl1 CLIP tag distribution in WT FVB quadriceps muscle. (B) Mbnl-regulated alternative polyadenylation patterns in vivo. Wiggle plots of Mbnl1 HITS-CLIP (dark blue) together with PolyA-seq of WT adult (light blue), WT P1 (green) and HSA^LR adult (red) muscle. (C) Normalized Calm3 and Tuba4a distal/total (D/T) pA ratios were determined for WT B6 adult (dark blue), DKO (orange), WT FVB adult (light blue), WT P1 neonate (green), HSA^LR adult (red) and MBNL1 overexpression (MBNL1 OE, purple) mouse skeletal muscle (data represented as mean +/- SEM, *p < 0.05, **p < 0.01, ***p < 0.001). (D) Polyadenylation regulatory map comparing MEF Mbnl1-3 and HSA^LR patterns.

Figure 6. Dysregulation of Alternative Polyadenylation in DM1

(A) Predominance of proximal pA shifts in DM1 muscle. Scatter plots illustrating 3′ UTR shortening (proximal, red, n=1863) and lengthening (distal, blue, n=1425) in DM1 versus control muscle based on FDR < 0.05 and −0.15>dI>0.15 (no shift, n=20580). (B) PolyA-seq revealed a proximal to distal pA shift for the ncRNA that modulates transcription of the DHFR coding transcript. A diagram of the DHFR coding (major) and non-coding (ncRNA, minor, dash line) transcriptional units (black box, coding region; thick black line, 3′ UTR) are illustrated together with PolyA-seq wiggle plots of control (blue) and DM1 (red). Arrows indicated the site of transcription initiation and only four exons are included for the major transcript. (C) Concordance between pA location and RNA level. RNA levels (relative to WT) were determined by qRT-PCR for DHFR (DHFR, DHFRnc) as well as genes involved in the mTOR (FOXO1, HDAC5, RPTOR) and ubiquitination (USP14) pathways (data represented as mean +/- SEM, *p < 0.05, **p < 0.01). (D) GO analysis based on PolyA-seq analysis of the principle biochemical pathways affected by APA changes in DM1. (E) PolyA-seq wiggle plots of control (blue) and DM1 (red) of RPTOR and HDAC5 terminal exons (black box, coding region; thick black line, 3′ UTR). Also indicated are the positions of potential miR binding sites (UCSC Genome Browser).

Figure 7. AllExon Microarray Analysis of APA in DM1 and DM2 muscle

(A) AllExon array hybridization results for probe sets representing PDLIM5 (top) and DNAJB6 (bottom) for muscle isolated from healthy individuals (blue line, N), DM1 (red), DM2 (green) and FSHD (FSH, violet) patients. Probe sets showing significant differences for both DM1 vs N and DM2 vs N comparisons are indicated by orange frames. Positions of differentially expressed RNA regions are marked on gene maps with experimentally confirmed polyA sites indicated (pA1, pA2; black box, coding region; thick black line, 3′ UTR; thin line, introns). The positions of RT-PCR primers (proximal, distal) are shown by arrows below each gene structure. Also shown are APA-specific RT-PCR gels that confirmed the transition from proximal (P) to distal (D) for PDLIM5, and from distal to proximal for DNAJB6, polyA sites in DM1 versus normal adult muscle. (B) Both Pdlim5 and Dnajb6 showed pA shifts during mouse muscle development from embryonic day (E)18, postnatal days P2, P8 and P20 to adult (Ad) mice. Note the fetal (E18) and neonatal (P2) patterns are similar to DM1. (C) Model for MBNL control of alternative polyadenylation. When MBNL protein (blue ovals) binding sites overlap a polyA site, they repress recruitment of 3′ end processing factors (3′ PF) while binding primarily upstream activates 3′ end processing at the downstream site. Depletion of MBNL activity due to sequestration by CUG^exp RNAs results in aberrant activation of polyA sites normally expressed during embryogenesis (red pA) and silencing of adult polyA sites (blue pA).