Mechanistic determinants of MBNL activity

Abstract

Muscleblind-like (MBNL) proteins are critical RNA processing factors in development. MBNL activity is disrupted in the neuromuscular disease myotonic dystrophy type 1 (DM1), due to the instability of a non-coding microsatellite in the DMPK gene and the expression of CUG expansion (CUG^exp) RNAs. Pathogenic interactions between MBNL and CUG^exp RNA lead to the formation of nuclear complexes termed foci and prevent MBNL function in pre-mRNA processing. The existence of multiple MBNL genes, as well as multiple protein isoforms, raises the question of whether different MBNL proteins possess unique or redundant functions. To address this question, we coexpressed three MBNL paralogs in cells at equivalent levels and characterized both specific and redundant roles of these proteins in alternative splicing and RNA foci dynamics. When coexpressed in the same cells, MBNL1, MBNL2 and MBNL3 bind the same RNA motifs with different affinities. While MBNL1 demonstrated the highest splicing activity, MBNL3 showed the lowest. When forming RNA foci, MBNL1 is the most mobile paralog, while MBNL3 is rather static and the most densely packed on CUG^exp RNA. Therefore, our results demonstrate that MBNL paralogs and gene-specific isoforms possess inherent functional differences, an outcome that could be enlisted to improve therapeutic strategies for DM1.

Figure 1.

Distinct splicing and expression patterns for MBNL paralogs. (A) Scheme of 10 expression constructs containing sequences of fluorescence proteins (GFP, mCherry or Dendra2) and MBNL1, MBNL2 and MBNL3 paralogs possessing or lacking three alternative exons (ex.54nt, ex.36nt, ex.95nt). For details on MBNL exonic composition see Supplementary Table S1. Constructs for comparable isoforms having the same alternative exons are divided into Set I, Set II and Set III. (B) Splicing profiles for MBNL ex.54nt, ex.36nt and ex.95nt in human tissues analyzed by RT-PCR. Dots represent PSI values. The adult and fetal samples from particular tissue types are indicated by a bar. Splicing results obtained for non-DM (N = 7), DM1 (N = 5) and DM2 (N = 7) muscle samples. Statistical significance was determined by the Student's t-test (∗ for P < 0.05, ∗∗ for P < 0.01 and ∗∗∗ for P < 0.001). (C) Relative expression level of exogenous MBNL paralogs. Exogenous proteins were detected by anti-GFP antibody and normalized to GAPDH. Bars represent average expression level and standard deviations are from two independent biological experiments. Statistical significance was determined by the Student's t-test (NS for P ≥ 0.05).

Figure 2.

MBNL1 is the strongest, while MBNL3 is the weakest, AS regulator. (A) Comparison of the relative mean for exOn/exOff splicing changes (|ΔPSI|) of 38 endogenous AS events in HeLa cells transfected with 10 different MBNL isoforms (left chart). Global analysis of exOn and exOff changes in C2C12 with silencing of MBNL determined by RNA-seq (central chart) (6) and DM2 skeletal muscles analyzed by microarrays (right chart) (53). For all analyses, the fold change was used as a measure of splicing strength for the indicated number of AS events. The statistical significance was determined by the Mann-Whitney U test. All comparisons revealed higher average changes for exOff than exOn. For more examples see Supplementary Figure S3. (B) Combined analysis of splicing changes (expressed as |ΔPSI|) of 38 tested AS events induced by MBNL paralogs from Set I, Set II and Set III. Statistical significance was assessed by Wilcoxon signed rank test (∗ for P <0.05, ∗∗ for P < 0.01 and ∗∗∗ for P < 0.001). (C) Differences in the regulation of exogenous mouse Atp2a1 ex.22 and human TNNT2 ex.4 by MBNL paralogs. The represented mean and standard deviation come from two biological replicas. Statistical significance was determined by the Student's t-test (∗ for P < 0.05, ∗∗ for P < 0.01 and ∗∗∗ for P < 0.001). For MBNL dose dependent changes, see Supplementary Figure S4.

Figure 3.

MBNL binding to RNA targets. (A) An example of overlapping MBNL1, MBNL2 and MBNL3 specific CLIP-seq clusters located in the region of intron 9 and alternative ex.10 (known also as ex.5; Supplementary Figure S1A) of mouse Mbnl1 based on the MBNL Interactome Browser (MIB.amu.edu.pl). For additional AS examples, see Supplementary Table S2. The presence of overlaid reads was detected for 81, 21 and 71% of AS events for MBNL1, MBNL2 and MBNL3, respectively. All YGCY sequence motifs located in this region, as well as evolutionary conservation are also shown. Note that Multi MIB also contains crosslinking induced mutations (CIMs), MBNL1-RIP-seq and CLIP-seq results for CELF1 (8) and NOVA (58) proteins. (B) A 10-mer sequence motif enrichment analysis from MBNL1, MBNL2 and MBNL3 CLIP-seq data determined by HOMER software. (C) The comparison of binding affinity of MBNL paralogs for 10 RNA fragments, indicating the strongest binding properties of MBNL1 and about 3-fold weaker of the other paralogs. However, in three examined instances, the binding affinity of all paralogs was alike. The dissociation constants (K_d) for MBNL1 constituted the normalization values. (D) MBNL paralogs have slightly different affinities for the 145 nt RNA fragment of Atp2a1 intron 22 in vitro. The K_d values significantly increase after blocking the MBNL1-specific binding site by 21nt-long antisense oligonucleotide (+AON) compared to control experiments (−AON). Dots represent the average percent of MBNL bound ± SD from two technical repetitions. For Mbnl1 exon 3, see Supplementary Figure S6C. For more details, see Supplementary Material & Methods. (E) Point mutations in crucial YGCY motifs compromise MBNL1, MBNL2 and MBNL3 binding efficiency in vitro. (Left panel) Nucleotide sequences of wild-type (wt) and mutated (mut#1-3) Calm3 3′UTR fragment and Mbnl2 intron 8/exon9 RNA fragment containing several YGCY motifs. (Right panel) Fold change of K_d values normalized to wt samples. (F) Removing the 111 nt sequence having the MBNL1-binding site in the Atp2a1 minigene (mut) also vastly affects MBNL2 and MBNL3 activity. (G) Blocking the MBNL-specific binding site in intron 22 of Atp2a1 minigene using AON reduces ex.22 inclusion for three MBNL paralogs in HeLa cells compared to the control AON (Ctrl.). Analogs results are represented for AONs designed against two other MBNL-binding sequences in Nfix and Ldb3. (F and G) Bars represent average PSI ± SD from two independent experiments and statistical significance was determined by the Student's t-test (∗ for P < 0.05, ∗∗ for P < 0.01 and ∗∗∗ for P < 0.001).

Figure 4.

Distinct localization patterns for MBNL paralogs. (A) The percentage of nuclear signal for GFP-MBNL fusions belonging to Set I, Set II and Set III determined by quantitative confocal microscopy analysis of the GFP fluorescence signal. Bars represent the mean from the whole volume of about 50 cells. Statistical significance was assessed by the Student's t-test (NS for P ≥ 0.05 and ∗∗∗ for P < 0.001). (B) Distinct nucleoplasmic and cytoplasmic distribution patterns for isoform pairs visualized by coexpression of MBNL proteins fused with GFP and mCherry (Set I and Set II). Isoforms having ex.54nt localize exclusively to the nucleus (Set III). (C) The Pearson correlation coefficient of relative nuclear concentration (normalized MBNLs expression level × relative nucleoplasmic distribution) with mean strength of 38 AS event changes (log|ΔPSI|) for six MBNL paralogs without alternative ex.54nt.

Figure 5.

Specific MBNL alternative exons modulate splicing activity. (A) Compared splicing activity (expressed as PSI values) of all tested MBNL proteins for four selected AS events analyzed by RT-PCR. Bars represent average PSI ± SD from three independent experiments and statistical significance was determined by the Student's t-test (∗ for P < 0.05, ∗∗ for P < 0.01 and ∗∗∗ for P < 0.001). White stars refer to statistical differences between a particular MBNL isoform and GFP control. Black stars refer to statistical differences between comparable MBNL isoforms having or lacking a sequence encoded by alternative ex.54nt, ex.36nt and ex.95nt. Supplementary Table S2 contains results for all 38 AS events. (B) Combined analysis of the 38 tested AS events for the comparable MBNL isoforms differing in the presence or absence of sequences encoded by ex.54nt, ex.36nt or ex.95nt. The comparison was made for nine pairs of isoforms, four for ex.54nt, three for ex.36nt and two for ex.95nt. AS events were divided into categories showing stronger, weaker or no splicing changes for individual isoform pairs taking statistical significance into consideration (‘no change’, P ≥ 0.05; weaker and stronger, P < 0.05; Student's t-test). Note that the mean strength of splicing changes for isoforms having ex.95nt was 2.6-time higher for exons under positive control compared to ex.36nt. The pie charts represent percentage of exOns and exOffs for each category represent (more information in text) (C) The influence of ex.54nt and ex.36nt on the splicing regulation of exogenous mouse Atp2a1 ex.22 and human TNNT2 ex.4. Bars represent average PSI ± SD from three independent experiments and statistical significance was determined by the Student's t-test (∗ for P < 0.05, ∗∗ for P < 0.01 and ∗∗∗ for P < 0.001). (D) Differences in MBNL1 isoform binding site distribution determined by CLIP-seq analysis. Percentage of unique CLIP-seq reads mapped to different regions of transcripts, namely introns located only in pre-mRNA (nucleus) and coding sequences (CDS), 5′UTRs and 3′UTRs located in both pre-mRNA and mRNA (nucleus and cytoplasm) for three MBNL1 isoforms (MB1-40 and MB1-41 without ex.54nt and MB1-43 with ex.54nt showing exclusively nuclear localization). Pink arrows indicate differences between percentage of reads for MBNL1 isoforms with and without ex.54nt.

Figure 6.

MBNL on CUG^exp RNA foci. (A) High affinity of recombinant MBNL1, MBNL2 and MBNL3 for (CUG)20 RNA in vitro. The K_d values are indicated. For (CCUG)14 and (CAG)20 transcripts, see Supplementary Figure S6E. (B) Overexpression of MBNL paralogs significantly increase CUG^exp foci volume (‘foci pumping’) measured by FISH for ∼130 foci. There is no difference in CUG^exp foci volume distribution between cells transfected with GFP and without protein overexpression (no ovex.). Statistical significance was assessed by Mann-Whitney U test (∗∗ for P < 0.01 and ∗∗∗ for P < 0.001). (C) Correlation between RNA foci volume with MBNL volume (measured by the GFP signal). The Spearman's ρ is between 0.87 and 0.95. FISH and GFP fluorescence measurements were performed using quantitative confocal microscopy, see Supplementary Figure S9. (D) Analysis of FRET efficiency (E) between GFP and mCherry either fused together (left picture) or with two MBNL proteins or with CELF1 in a control experiment (right picture) in the absence or presence of a CUG^exp transcript. The E-values which are average from 10-20 analyses for pairs of full length MB1-41 and truncated MB1-C in CUG^exp foci are as high as for a positive GFP-mCherry control in an entire cell. In the absence of CUG^exp, the E-value is slightly above the background and similar to the value observed for a CELF1 and MB1-41 pair. For more results, see Supplementary Figure S10. (E) MBNL paralogs differ in E. Statistical significance was determined by Student's t-test (NS for P ≥ 0.05, ∗∗ for P < 0.01). (F) MBNL paralogs differ in their mobile fraction in FRAP experiments performed in cells saturated with MBNL protein. The results are mean from about 20 nuclei ± SD and statistical significance was determined by Mann-Whitney U test (∗ for P < 0.05 and ∗∗∗ for P < 0.001). See Supplementary Video S3. (G) The presence of a sequence encoded by alternative ex.95nt reduces MBNL2 mobility in FRAP experiments. For more examples, see Supplementary Figure S11A. (H) Photoswitching of Dendra2 fused with MB1-41 in cells with saturated levels of MBNL protein. Photoconverted (red, emission 573 nm) protein is shifting away whereas unconverted (green, emission 507 nm) protein is associating with CUG^exp foci (see Supplementary Video S4). The same analysis for MB1-40 was shown in Supplementary Figure S11B. (I) There is no correlation between the relative nuclear fluorescence intensity of GFP fused MBNL proteins and the mobile MBNL fraction. (J) FRAP experiment for MB2-38 in cells with the very low fluorescence signal of the fusion protein in nuclei. Note that the mobile fraction is three-times lower in the unsaturated compared to saturated state of MBNL (see Supplementary Figure S11A for comparison). (K) The diffusion of photoconverted Dendra2 fused with MBNL1 within individual CUG^exp focus in a cell with the low fluorescence signal in nucleoplasm (unsaturated MBNL state). Quantification was performed from two regions of interest (ROI1 and ROI2). The ROI1 is an area that was photoconverted by a laser and the red signal decreases during the course of time. On the other hand, in the distant ROI2 of the same CUG^exp focus in which only green signal was detected in time 0, the red signal from photoconverted Dendra2 increases for several seconds. In this situation, MBNL proteins are moving to other binding sites on CUG^exp RNA and are not dissociating from the foci.

Figure 7.

Intracellular determinants of MBNL activity and localization. (A) The three main determinants of MBNL activity determined in current study. (B) Deterministic nature of MBNL-CUG^exp foci formation is disrupted by the chaos within individual focus. Two distinct stages, differing in the number of MBNL-binding sites on CUG^exp were shown. MBNLs would be in saturated (left panel) or unsaturated state (right panel) in CUG^exp foci. The latter enables efficient MBNL sequestration. See the text for more details.