MBNL splicing activity depends on RNA binding site structural context

Abstract

Muscleblind-like (MBNL) proteins are conserved RNA-binding factors involved in alternative splicing (AS) regulation during development. While AS is controlled by distribution of MBNL paralogs and isoforms, the affinity of these proteins for specific RNA-binding regions and their location within transcripts, it is currently unclear how RNA structure impacts MBNL-mediated AS regulation. Here, we defined the RNA structural determinants affecting MBNL-dependent AS activity using both cellular and biochemical assays. While enhanced inclusion of MBNL-regulated alternative exons is controlled by the arrangement and number of MBNL binding sites within unstructured RNA, when these sites are embedded in a RNA hairpin MBNL binds preferentially to one side of stem region. Surprisingly, binding of MBNL proteins to RNA targets did not entirely correlate with AS efficiency. Moreover, comparison of MBNL proteins revealed structure-dependent competitive behavior between the paralogs. Our results showed that the structure of targeted RNAs is a prevalent component of the mechanism of alternative splicing regulation by MBNLs.

Figure 1.

Different structures of the same RNA binding site affect MBNL1 binding and splicing activity. (A) Proposed secondary structures of variants of the Pphln1 transcript fragment with previously defined functional MBNL1-binding motifs marked in green and in bold (44). The optimal thermodynamic stability of YGCY-containing RNA regions is expressed in Gibbs free energy (ΔG) in kcal/mol for the reaction at 37°C using RNAfold software (46) and is marked in blue. T2 and S1 nucleases-specific cleavage sites and cleavage intensities are specified for each probe according to symbols shown in the legend. Unique nucleotides, not shared between RNA variants, are marked in grey. See Figure S1 for RNA structures of other transcript variants. (B) A simplified scheme of a biochemical assay based on double membrane filtration. The radioactivity on the upper nitrocellulose film (representing ³²P-RNA bound to rMBNL1) and that on a lower nylon film (representing free ³²P-RNA) was quantified for multiple protein concentrations and MBNL1 affinity (K_d value) for each transcript was calculated (see Figure S2 for more details). (C) The quantification of the biochemical assay showing different rMBNL1 binding affinities to two structural variants of three natural transcript fragments. The results are mean K_d values; n = 2 for each protein concentration (in the range of 0–250 nM of rMBNL1). (D) A model of a hybrid Atp2a1 minigene with the Atp2a1 gene fragment between exons 21 and 23 and with a natural MBNL-binding site within intron 22 (I22) in pre-mRNA (Atp2a1-WT) substituted with different YGCY motif-containing RNA fragments. These RNA fragments serve as artificial regulatory elements to test MBNL activity depending on RNA structural organization and YGCY arrangement. A stable helical region formed by Atp2a1 I22, restriction sites used for cloning and a 5-bp-long artificial sequence located at the base of an insert allowing the preservation of its experimentally and in silico-defined RNA secondary structure are marked with a blue box (see also Figure S2b). Alternative and constitutive exons are marked with white and grey boxes, respectively; E, exon. (E) Different efficiencies of alternative E22 inclusion into the mRNA of hybrid Atp2a1 minigenes depending on the studied RNA regulatory elements upon eGFP-control or MBNL1 overexpression (OE) in HeLa cells; n = 3.

Figure 2.

MBNL1 binding and splicing activity is regulated by both the number of UGCU motifs and structural context. (A) RNA secondary structures of semi-stable (ds; double-stranded) and unstructured (ss; single-stranded) artificial regulatory elements which are base paired at the 5′ and 3′ ends and internally (only ds RNA) to force the formation of one thermodynamically optimal secondary structure confirmed by us in several RNA structure prediction software programs (46–48). MBNL-binding motifs are numbered from 5′-end and marked in green. The RNA names correspond to the number of UGCU motifs (2, 3 or 4) and the structure type (ds or ss). The splicing response of pre-mRNAs of hybrid minigenes with ds or ss RNAs incorporated in I22 upon (B) endogenous level of MBNLs or (C) MBNL1 overexpression (OE) in HeLa cells. Vertical asterisks denote the statistical significance of results in comparison to a control experiment (Ctrl-Δ; transfection with Atp1a1-Δ minigene). Note a positive effect of the increasing UGCU motif number on alternative splicing - the PSI of E22 for 2ss < 3ss < 4ss; MBNL1 dose-dependency of weaker regulatory elements - the PSI of E22 for 2ds < 2ss > 3ds; and the structural organization of an RNA regulatory element surpassing the number of UGCU motifs - PSI of E22 for 4ds = 3ss; 2ss > 3ds; n = 2. (D) Quantification of the biochemical assay showing relative rMBNL1 binding affinity for RNA fragments normalized to K_d for the 4ds RNA molecule; n = 2 for each protein concentration (in the range of 0–200 nM of rMBNL1). (E) RT-PCR showing the splicing response of hybrid Atp2a1 minigenes representing ss and ds groups of RNAs with or without MBNL1 OE in Mbnl1; Mbnl2 DKO MEFs. The asterisks denote the statistical significance of results compared to cells treated with control eGFP construct (Ctrl). Ctrl-Δ, transfection with Atp1a1-Δ minigene; n = 3.

Figure 3.

Distance between UGCU motifs modulates MBNL-mediated splicing regulation but not binding affinity. (A) The structures of ss RNA regulatory elements with distinctly interspaced UGCU motifs marked in green. The RNA names correspond to the distance between consecutive UGCU motifs, which is marked in brackets surrounded by the number of adjacent motifs. (B) As in Figure 2D but for a group of ss RNAs with interspaced UGCU motifs; n = 2–4 for each protein concentration (in the range of 0–400 nM of rMBNL1). (C) As in Figure 2C but for a group of ss RNAs with interspaced UGCU motifs; n = 2. Note no or very weak regulatory properties of RNAs with overlapping motifs - 2ss(–1), 3ss(–1) and 4ss(–1). RNAs with sequential motifs - 4ss(0), 3ss(0) and 2ss(0), exceeded all other tested regulatory elements within the UGCU number sets. UGCU separation had an adverse effect on E22 inclusion - the PSI of E22 for 4ss(0) > 4ss3(5)1 = 4ss2(5)2 > 4ss(5). Note the redundant impact of UGCU number - PSI of E22 for 3ss2(5)1 = 4ss(5). Vertical asterisks denote the statistical significance of results in comparison to a control experiment (Ctrl-Δ; transfection with Atp1a1-Δ minigene). (D) The splicing response of hybrid Atp2a1 minigenes with regulatory elements distinguished by either overlapping or sequential arrangement of distinct number of UGCU motifs. The graph shows RT-PCR results for DKO MEFs co-transfected with hybrid minigenes and the MBNL1 expression vector. Vertical asterisks denote the statistical significance of results in comparison to Ctrl-Δ construct; n = 3.

Figure 4.

Effect of RNA structural context on MBNL splicing activity. (A) The RNA secondary structures of ds RNAs containing four UGCU motifs marked in green. Dashed blue lines depict a reciprocal position of UGCU motifs. Orange rectangles depict ds helical regions stabilizing the RNA secondary structure. The RNA names correspond to the distance between consecutive UGCU motifs which is marked in brackets surrounded by the number of adjacent motifs. (B and C) As in Figure 2D, but for a group of ds RNAs; n = 2–4 for each protein concentration (in the range of 0–200 nM of rMBNL1). (D and E) As in Figure 2C but for a group of ds RNAs; n = 2. Note the decreasing E22 inclusion upon separating tandems of motifs - the PSI of E22 for 4ds2(6)2 > 4ds2(12)2 > 4ds2(24)2. Vertical and horizontal asterisks denote the statistical significance of results in comparison to Ctrl-Δ and 4ds constructs, respectively. (F) The Pearson correlation coefficient of rMBNL1 binding affinity (K_d; nM) and splicing activity (PSI values) upon endogenous level of MBNLs for 20 artificial RNA structures analyzed in this study (based on the results shown in Figures 2–4). The RNAs represent two groups of structures (ss or ds) with the same sequence background which showed no effect on E22 inclusion in Mbnl1; Mbnl2 DKO cells (Figure 2E). Dashed lines represent the 95% confidence interval.

Figure 5.

Natural RNA structural determinants that modulate MBNL activity. (A) A secondary structure of Calm3-RNA containing MBNL1-binding motifs (HGCH – H stands for U, C or A) marked in green and numbered. Point mutations within the motifs are marked with orange circles. The structural modifications of particular Calm3-RNA regions are presented in boxes. Substitutions and insertions or deletions are marked with red dashed circles or triangles, respectively. (B) RT-PCR results showing the splicing response of pre-mRNAs of hybrid minigenes with incorporated different Calm3 regulatory elements carrying HGCH mutations or structural modifications upon MBNL1, MBNL2 and MBNL3 OE in HeLa cells. The MBNL paralogs are identical with respect to splicing isoforms (lacking residues encoded by alternative exons 5 and 7); n = 2. Vertical and horizontal asterisks denote the statistical significance of results in comparison to Ctrl-Δ and Calm3 WT constructs, respectively. Ctrl-Δ, Atp2a1-Δ minigene. (C) Quantification of the biochemical assay showing binding affinity of recombinant rMBNL1, rMBNL2 and rMBNL3 to intact Calm3-RNA, HGCH motif and RNA secondary structure mutants normalized to Calm3-RNA WT; n = 2–4 for each protein concentration (in the range of 0–200 nM of rMBNL1). Vertical asterisks denote the statistical significance of results in comparison to intact Calm3-RNA.

Figure 6.

Competition between MBNL proteins is driven by RNA regulatory element organization. (A) RNA-seq read coverage across two MBNL-dependent alternative exons indicating their adult-like splicing profiles in Mbnl3 knockout (3KO) myoblasts compared to wild-type cells and Mbnl1; Mbnl2 DKO quadriceps muscle (postnatal, day 0); alternative and constitutive exons are marked with white or gray boxes, respectively; n = 2. Note that the effect of 3KO is opposite to the effect of DKO. (B) RT-PCR results of splicing changes in two MBNL1-dependent alternative exons which direction was opposite upon MBNL3 silencing (3KD) compared to MBNL1; MBNL2 double knockdown (DKD) with respect to cells treated with control siRNA in human skeletal myoblasts (HSkM); n = 3. (C) RT-PCR results of splicing changes in MBNL1-dependent alternative exons which direction was similar upon MBNL3 and MBNL1 OE in Mbnl1; Mbnl2 DKO MEFs; Ctrl, the results from eGFP construct transfected cells (marked in grey); n = 3. (D) A competition assay between MBNL1 and MBNL3 paralogs. RT-PCR results showing the splicing response of pre-mRNAs with distinctly arranged UGCU motifs in intronic regulatory elements in constant background of MBNL1 OE (marked in orange) and with increasing concentration of MBNL3 (marked in blue) in HeLa cells. MBNL1 and MBNL3 paralogs are identical with respect to splicing isoforms (lacking residues encoded by alternative exons 5 and 7); Ctrl, the results from eGFP construct transfected cells (marked in black). The asterisks denote the statistical significance of results in comparison to cells with MBNL1 OE only (marked in orange); n = 2. Each graph represents the results obtained for two constructs belonging to the category of RNA regulatory elements with overlapping, sequential or interspaced UGCU motifs. (E) Image of polyacrylamide gels presenting the expression level of eGFP-MBNL1 and eGFP-MBNL3 fusion proteins and eGFP alone. After electrophoresis gels were scanned for eGFP fluorescence (protein samples were not heat-denatured before gel loading). (F) A schematic representation of MBNL1 splicing isoforms differing in the presence of residues encoded by alternative exons 5 and 7 (marked in brown and beige, respectively) but sharing the same RNA binding domain composed of four ZFs (marked in green). (G) As in Figure 6D but for MBNL1 isoform pairs. An equal amount of applied expression vectors resulted in 2:1 ratio of expressed proteins for MBNL1–42 versus MBNL1–40/41 (30). MBNL1–40, MBNL1–41 and MBNL1–42 are marked in orange, beige and brown, respectively. The asterisks denote the statistical significance of results in comparison to cells with overexpression of only one isoform; n = 2.

Figure 7.

RNA regulatory element organization influences the outcome of MBNL-determined AS profile. (A) Upon MBNL binding to distinctly arranged or structured RNA motifs, changes in availability or conformation of splice-activation elements of the protein may occur and lead to alterations in the strength of the MBNL interaction with other splicing factors including U2 auxiliary factor 65 kDa subunit (U2AF65), resulting in weakened or enhanced alternative CE inclusion (depicted by the size of green arrows). (B) A model in which MBNL1 and MBNL3 compete at a dose- and RNA substrate-dependent manner during muscle development and regeneration as well as in pathological conditions (DM) modulating the AS profile; more explanation in the text. To simplify, MBNL splicing isoforms and MBNL2 paralog were omitted in the scheme.