MEF2C Directly Interacts with Pre-miRNAs and Distinct RNPs to Post-Transcriptionally Regulate miR-23a-miR-27a-miR-24-2 microRNA Cluster Member Expression

Transcriptional regulation constitutes a key step in gene expression regulation. Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS box family involved in the early development of several cell types, including muscle cells. Over the last decade, a novel layer of complexity modulating gene regulation has emerged as non-coding RNAs have been identified, impacting both transcriptional and post-transcriptional regulation. microRNAs represent the most studied and abundantly expressed subtype of small non-coding RNAs, and their functional roles have been widely documented. On the other hand, our knowledge of the transcriptional and post-transcriptional regulatory mechanisms that drive microRNA expression is still incipient. We recently demonstrated that MEF2C is able to transactivate the long, but not short, regulatory element upstream of the miR-23a-miR-27a-miR-24-2 transcriptional start site. However, MEF2C over-expression and silencing, respectively, displayed distinct effects on each of the miR-23a-miR-27a-miR-24-2 mature cluster members without affecting pri-miRNA expression levels, thus supporting additional MEF2C-driven regulatory mechanisms. Within this study, we demonstrated a complex post-transcriptional regulatory mechanism directed by MEF2C in the regulation of miR-23a-miR-27a-miR-24-2 cluster members, distinctly involving different domains of the MEF2C transcription factor and the physical interaction with pre-miRNAs and Ksrp, HnRNPa3 and Ddx17 transcripts.


Introduction
Transcriptional regulation constitutes a key step in gene expression regulation.Multiple types of transcription factors have been identified from flies to humans, regulating multiple developmental, homeostatic and pathological processes [1][2][3].In this context, a core of transcription factors has been identified to play essential roles in myogenesis, such as SRF, NKX2.5, GATA4 and MEF2C [4].Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS box family involved in the early development of several cell types, including neural, immune, cartilaginous and endothelial cells, yet the main role of MEF2C is exerted by regulating muscle development (i.e., skeletal, cardiac and smooth) [5][6][7][8][9][10][11]. MEF2C-deficient mice are embryonically lethal, displaying complex cardiovascular defects as the early heart tube does not undergo looping morphogenesis, resulting in the absence of the future right ventricle [12,13].Notably, MEF2C also plays a pivotal role in cardiac pathological conditions such as cardiac hypertrophy [14], and it represents an essential cornerstone for cardiac reprogramming [15,16].
Over the last decade, a novel layer of complexity in gene regulation has emerged with the identification of non-coding RNAs, impacting both transcriptional and post-transcriptional processes.Non-coding RNAs are broadly classified according to their transcript length into small non-coding RNAs (<200 nt) and long non-coding RNAs (>200 nt) [30].Among the small non-coding RNAs, microRNAs represent the most studied and abundantly expressed subtype.MicroRNAs are nuclearly encoded and transcribed into microRNA precursor molecules by RNA polymerase II.In certain genomic locations, microRNAs are clustered, resulting in a primary transcript containing multiple microRNA precursors, leading to a pri-mRNA precursor.Pri-miRNAs are then processed by nucleases such as Drosha and Dgcr8 to generate distinct pre-miRNA molecules that are subsequently exported by the exportin-5/Ran protein complex to the cytoplasm [31].Within the cytoplasm, the pre-miRNAs are further processed into mature microRNA duplex by Dicer RNAse and loaded into the RISC complex.Within the RISC complex, one strand of the double-stranded microRNA molecule is degraded, leaving the mature microRNA to scan RNA molecules for the sequence homology of its seed sequence, leading to post-transcriptional RNA target cleavage, translation repression and/or RNA deadenylation.As a result, in most cases, the abundance of the miRNA/protein target decreases [32].Importantly, emerging evidence suggests that certain microRNAs can also modulate transcriptional regulation by exerting their function within the nucleus, thus impacting alternative splicing and RNA and microRNA transcriptional regulation [33].
Lee et al. [65] demonstrated that the miR-23a-miR-27a-miR-24-2 cluster is transcribed as an RNA polymerase II-dependent primary transcript whose main transcriptional regulation is driven by a −600 bp upstream promoter.We subsequently reported the identification of upstream regulatory elements driving miR-23a-miR-27a-miR-24-2 transcriptional regulation in both cardiac and skeletal muscle cells [66].Within this context, we demonstrated that MEF2C is able to transactivate the long (−1830 to +1 nt) regulatory element but not the short (−776 to +1 nt) element, in accordance with the identification of MEF2 regulatory binding sites distribution.However, MEF2C over-expression and silencing, respectively, displayed distinct effects on each of the miR-23a-miR-27a-miR-24-2 cluster members with-out affecting pri-miRNA expression levels in different cell types [66], thus supporting additional MEF2C-driven regulatory mechanisms.Within this study, we report complex transcriptional and post-transcriptional regulatory mechanisms directed by MEF2C in the regulation of the miR-23a-miR-27a-miR-24-2 cluster, distinctly involving different domains of the MEF2C transcription factor and the physical interaction with pre-miRNAs and Ksrp, HnRNPa3 and Ddx17 transcripts.

Results
We have previously characterized the transcriptional potential of the 1.8 Kb upstream sequences of the miR-23a-miR-27a-miR-24-2 cluster and reported that MEF2C is capable of transcriptionally activating these regulatory regions in HL1 atrial cardiomyocytes.Such transcriptional activation thus enhances the expression of the miR-23a-miR-27a-miR-24-2 pri-miRNA.However, we have also previously reported that MEF2C over-expression and inhibition, respectively, distinctly regulate the expression of each of the miR-23a-miR-27a-miR-24-2 mature cluster members independently of its transcriptional potential.Notably, the modulation of miR-23a-miR-27a-miR-24-2 cluster members by MEF2C is tissue-specific.Therefore, our previous data suggest that MEF2C modulation of miR-23a-miR-27a-miR-24-2 cluster members is exerted by either direct or indirect post-transcriptional mechanisms.Since microRNAs have been recently reported to exert both cytoplasmic and nuclear functions, we initially explored the subcellular location of the miR-23a-miR-27a-miR-24-2 mature cluster members.RT-qPCR of nuclear and cytoplasmic fractions revealed that all three members, i.e., miR-23a_3p, miR-27a_3p and miR-24_3p, are similarly localized in both subcellular compartments in HL1 cardiomyocytes (Figure 1A), in contrast to miR-130a, which is preferentially and significantly enhanced in the cytoplasm (Figure 1A), while Xist2 is enhanced in the nucleus (Figure 1A), serving as internal subcellular fractioning control.
We subsequently tested which part of the MEF2C transcription factor exerts pre-miR-23a and pre-miR-27a modulation and whether it also affects the expression of the mature microRNA cluster members.For this purpose, we constructed two distinct MEF2C variants.The first one lacks the SRF-type DNA-binding and dimerization domain, the MADS_MEF2_like and the HJURP_C domain at the 5' end (MEF2C 5 ′ del) (Supplementary Figure S1B).The second variant lacks the 3' end (MEF2C 3 ′ del) while maintaining these domains (Supplementary Figure S1B).In addition, we also performed MEF2C over-expression and silencing studies, achieving successful modification of MEF2C expression levels.In addition, the overexpression of MEF2C full-length, MEF2C 5 ′ del and MEF2C 3 ′ del showed increased levels, both at the transcript and protein levels, as compared to non-transfected controls, respectively (Supplementary Figure S1C,D).Furthermore, transactivation assays of the L regulatory element of the miR-23a-miR-27a-miR-24-2 locus were successfully achieved with MEF2C full-length and MEF2C 3 ′ del constructs but not with MEF2C 5 ′ del, as expected, since the latter lacks DNA-binding and dimerization domains (Supplementary Figure S1E).Note that all three mi-croRNAs are similarly expressed in the nucleus and cytoplasm in contrast to miR-130a, which is primarily cytoplasmic, and the long non-coding RNA Xist2, which is preferentially nuclear.Panel (B) RT-qPCR analyses of Mef2c pulldown assays for pre-miR-23a, pre-miR-27a and pre-miR-24, respectively.Note that increased levels are observed for pre-miR-23a and pre-miR-27a but not for pre-miR-24.Panel (C) RT-qPCR analyses of Mef2c pulldown assays for mature miR-23a_3p, miR-27a_3p and miR-24_3p, respectively.Note that none of the mature microRNAs are increased after Mef2c pulldown assays.Panel (D) RT-qPCR analyses of Mef2c pulldown assays for pri-miR-23-miR-27a-miR-24-2.Panel (E) Schematic representation of the Mef2c association with the miR-23a-miR-27a-Figure 1. Panel (A) RT-qPCR analyses of the nuclear and cytoplasmic distribution of miR-23a_3p, miR-27a_3p and miR-24_3p mature microRNAs in HL1 cardiomyocytes.Note that all three microRNAs are similarly expressed in the nucleus and cytoplasm in contrast to miR-130a, which is primarily cytoplasmic, and the long non-coding RNA Xist2, which is preferentially nuclear.Panel (B) RT-qPCR analyses of Mef2c pulldown assays for pre-miR-23a, pre-miR-27a and pre-miR-24, respectively.Note that increased levels are observed for pre-miR-23a and pre-miR-27a but not for pre-miR-24.Panel (C) RT-qPCR analyses of Mef2c pulldown assays for mature miR-23a_3p, miR-27a_3p and miR-24_3p, respectively.Note that none of the mature microRNAs are increased after Mef2c pulldown assays.Panel (D) RT-qPCR analyses of Mef2c pulldown assays for pri-miR-23-miR-27a-miR-24-2.Panel (E) Schematic representation of the Mef2c association with the miR-23a-miR-27a-miR-24-2 clustered microRNAs.All data are normalized to Gapdh for mRNA expression analyses and to 5S for microRNA expression analyses.* p < 0.05, ** p < 0.01, *** p < 0.001.
Adar1, Ddx5 and HnRNPa1 displayed increased expression in 3T3 fibroblasts as compared to HL1 atrial cardiomyocytes and Sol8 skeletal myoblasts (Figure 3A).Ksrp displayed similarly enhanced expression in 3T3 fibroblasts and HL1 atrial cardiomyocytes as compared to Sol8 skeletal myoblasts (Figure 3A).HnRNPa2b1 and Ddx17 display a similar expression pattern with enhanced expression in HL1 cardiomyocytes, while HhRNPa3 displayed the opposite pattern, i.e., decreased expression in HL1 atrial cardiomyocytes as compared to 3T3 fibroblasts and Sol8 skeletal myoblasts (Figure 3A).Overall, these data showed that the RNA constituents of all mentioned RNPs are expressed in these three distinct cell lines tested.However, the distinct RNPs showed a differential expression in these cell lines, thus supporting the plausible contribution of these RNPs in regulating the distinct miR-23a-miR-27a-miR-24-2 cluster members by MEF2C in different cell types, as previously demonstrated [64].
We also tested whether these RNP transcripts are distinctly distributed within the subcellular compartments in HL1 cardiomyocytes.Our data revealed that Adar1 is highly enriched in the nuclear compartment, whereas Ddx5, Ddx17 and Ksrp are prominently localized in the cytoplasm.On the other hand, HnRNPa1, HnRNPa3 and HnRNPa2b1 are similarly distributed within both nuclear and cytoplasmic compartments, in line with MEF2C mRNA distribution (Figure 3B).

Discussion
Within the last decade, our understanding of the functional role of distinct mi-croRNAs has greatly emerged; however, our knowledge of the transcriptional and posttranscriptional regulatory mechanisms driving microRNA expression is still incipient.We previously demonstrated that MEF2C over-expression and silencing, respectively, displayed distinct effects on each of the mature miR-23a-miR-27a-miR-24-2 cluster members [66], thus supporting additional MEF2C-driven regulatory mechanisms.We provide herein evidence that MEF2C can directly bind to pre-miR23a and pre-miR-27a but not to pre-miR-24-2.Importantly, MEF2C does not directly bind to either the pri-miRNA miR-23a-miR-27a-24-2 precursor or to the mature miR-23a_3p, miR-27a_3p and miR-24_3p molecules.Furthermore, we also demonstrated that distinct MEF2C domains can differentially modulate both pre-miRNA and microRNA expression.While there is emerging evidence that distinct proteins can influence MEF2C expression levels, leading to sumoylation and caspase cleavage [26,71], this is, to the best of our knowledge, the first proof that a transcription factor can influence microRNA biogenesis by directly interacting with pre-miRNA molecules.
On the other hand, ample evidence is reported on the key role of distinct ribonucleoproteins (RNPs) in modulating microRNA expression [72][73][74][75][76][77].Thus, to further support the plausible role of several of these RNPs in MEF2C-driven miR-23a-miR-27a-miR-24-2 expression, we analyzed the expression of seven distinct RNP transcripts in three distinct cell types (fibroblasts, cardiomyocytes and skeletal muscle myoblasts), demonstrating that all of them are indeed expressed while displaying cell type enrichment, i.e., Ddx17 and HnRNPa2b1 are more abundantly expressed in cardiomyocytes, while Adar1, Ddx5 and HnRNPa3 are widely expressed in fibroblasts.Furthermore, we demonstrated that these RNP transcripts displayed distinct subcellular distribution patterns, i.e., Adar1 is primarily located in the nucleus, Ddx5, Ddx17 and Ksrp are primarily in the cytoplasm, while HnRNPa1, HnRNPa2b1 and HnRNPa3 are both nuclear and cytoplasmic, in line with previous reports [78][79][80][81][82][83].Importantly, we firstly demonstrated that mature miR-23a-miR-27a-miR-24-2 cluster microRNA members are equally distributed in both nuclear and cytoplasmic subcellular compartments, supporting the notion that they might exert distinct functional roles, as recently reported [84][85][86][87][88], and thus can be distinctly regulated in the cytoplasm vs. the nucleus.Furthermore, these data also support that distinct RNPs might be involved in the differential and subcellular compartment-specific expression of miR-23a-miR-27a-miR-24-2 cluster members.
Scarce evidence has been reported for transcription factors directly binding to RNPs [88], supporting their plausible role in post-transcriptional regulation.For MEF2C, only AUF1 binding has been reported, promoting skeletal muscle myogenesis [83].Within this study, we report for the first time that MEF2C can directly bind to Ddx17, HnRNPa3 and Ksrp mRNAs, respectively.Additionally, MEF2C indirectly regulates Adar1 and HnRNPa2b1 expression.Furthermore, we also demonstrate that distinct MEF2C domains differently contribute to RNP transcript expression.In this context, both 5 ′ and 3' MEF2C ends can selectively inhibit Ddx5, Ddx17 and Ksrp expression while enhancing Adar1 expression.On the other hand, HnRNPa1, HnRNPa3 and HnRNPa2b1 are distinctly regulated by MEF2C 3 ′ and 5' ends, respectively.While additional studies are required to fully understand the molecular mechanisms directing MEF2C 3 ′ and 5' ends modulation of these RNPs, our data support the notion that they might be transcriptionally regulated since the MEF2C 5 ′ del construct lacks transcriptional potential (Supplementary Figure S1D) and primarily downregulates their expression, while the MEF2C 3 ′ del construct displays the opposite pattern.In sum, our data demonstrate that MEF2C can directly and indirectly regulate distinct RNPs in cardiomyocytes, with a potential impact on miR-23a-miR-27a-miR-24-2 cluster member expression.
Overall, these data demonstrate the complex and pivotal role of distinct RNPs in regulating miR-23a-miR-27a-miR-24-2 cluster members and support the notion that distinct RNPs, particularly HnRNPa1 and Ksrp, play a pivotal role in regulating the differential expression of miR-23a-miR-27a-miR-24-2 cluster members by selectively acting on distinct pre-miRNAs.Surprisingly, the selective inhibition of mature miR-23a-miR-27a-miR-24-2 cluster members by RNP silencing is observed only for HnRNPa2b1 and HnRNPa3, but they do not recapitulate the effects provided by MEF2C silencing, supporting the notion that combinatorial rather than single MEF2C-driven RNP modulation is occurring.Furthermore, it is important to highlight in this context that all mature miR-23a-miR-27a-miR-24-2 cluster members are similarly expressed in both subcellular nuclear and cytoplasmic compartments as well as several RNPs.Notably, siRNA silencing would only be affecting those events occurring in the cytoplasm, and therefore, inhibition might only be partial.The causal relationship between such distinct subcellular compartment localization deserves further analysis and might provide novel insights into the precise molecular mechanisms controlling the differential expression of the mature microRNAs of genomic clustered microRNAs.
In summary, we provide herein evidence of the complex post-transcriptional regulatory mechanism exerted by MEF2C in the regulation of miR-23a-miR-27a-miR-24-2 cluster members (Figure 5).MEF2C can directly and selectively bind to pre-miR-23a_3p and pre-miR-27a_3p but not to pre-miR-24-2.Additionally, MEF2C can directly bind to distinct RNP transcripts, such as Ddx7, HhRNPa3 and Ksrp, while indirectly regulating the expression of other RNPs, such as Adar1 and HnRNPa2b1.Importantly, such regulation is distinctly exerted by the MEF2C amino-and carboxy-terminals.Silencing of MEF2C-binding RNP Ksrp selectively regulates pre-miR-23a and pre-miR-27a expression but not pre-miR-24-2, supporting the notion of a direct implication of this pathway on the differential expression of miR-23a-miR-27a-miR-24-2 cluster members, yet a combinatorial action of distinct RNPs seems to be required to fully achieve the final miR-23a-miR-27a-miR-24-2 cluster member expression of the mature microRNAs.In summary, we provide herein evidence of the complex post-transcriptional regulatory mechanism exerted by MEF2C in the regulation of miR-23a-miR-27a-miR-24-2 cluster members (Figure 5).MEF2C can directly and selectively bind to pre-miR-23a_3p and pre-miR-27a_3p but not to pre-miR-24-2.Additionally, MEF2C can directly bind to distinct RNP transcripts, such as Ddx7, HhRNPa3 and Ksrp, while indirectly regulating the expression of other RNPs, such as Adar1 and HnRNPa2b1.Importantly, such regulation is distinctly exerted by the MEF2C amino-and carboxy-terminals.Silencing of MEF2C-binding RNP Ksrp selectively regulates pre-miR-23a and pre-miR-27a expression but not pre-miR-24-2, supporting the notion of a direct implication of this pathway on the differential expression of miR-23a-miR-27a-miR-24-2 cluster members, yet a combinatorial action of distinct RNPs seems to be required to fully achieve the final miR-23a-miR-27a-miR-24-2 cluster member expression of the mature microRNAs.

MEF2C Pulldown Assays
For the immunoprecipitation of endogenous MEF2C, protein A-Sepharose beads (Abcam, Cambridge, UK) were coated with 15 µg of antibody that recognized MEF2C (#9792-Cell Signalling) or control IgG (Abcam, Cambridge, UK) overnight at 4 °C with  times after spinning down at 5000 g for 2 min at 4 • C. Protein complexes were incubated with 20 units of DNase I (15 min at 37 • C).In this step, an aliquot from each reaction was isolated for Western blot validation.Subsequently, they were further incubated with 0.1% SDS/0.5 mg/mL Proteinase K (30 min at 55 • C) with mixing to remove DNA and proteins, respectively, and centrifuged at 5000× g for 5 min to collect the supernatant.The RNA isolated from the IP materials (acid phenol-chloroform) was further assessed by RT-qRT-PCR analysis.

Nuclear/Cytoplasmic Distribution
Cytoplasmic and nuclear RNA fractions from HL1 cardiomyocytes were isolated with a Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Belmont, CA, USA) following the manufacturer's instructions.After RNA isolation, RT-qPCR analysis for nuclear enriched Xist2 mRNA marker and cytoplasmic Gapdh mRNA marker were performed to validate enrichment on each subcellular fraction.RT-qPCR analysis of distinct microRNAs, Xist2 and RNPs was subsequently performed, as detailed in the next sections.

RNA Isolation and Retrotranscription
Total RNA was isolated using the ReliaPrep RNA Cell Miniprep System (Promega, Madison, WI, USA), and DNase was treated using RNase-Free DNase according to the manufacturer's guidelines for 15 min at room temperature.In all cases, at least three distinct pooled samples were used to perform the corresponding RT-qPCR experiments.

RT-qPCR Analyses (mRNA)
First-strand cDNA was synthesized by using 100 ng of total RNA and a reverse transcription Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's guidelines.Negative controls to assess genomic contamination were performed for each sample, without reverse transcriptase, which resulted in all cases in no detectable amplification product.Real-time PCR experiments were performed with 2 µL of diluted cDNA, GoTaq qPCR Master Mix (Promega) and corresponding primer sets.Two internal controls, mouse Gusb and Gapdh mRNAs, were used in parallel for each run and represented as previously described [103][104][105].Amplification conditions were as follows: denaturalization step of 95  C for 10 min.All primers were designed to span exon-exon boundaries using the online Primer3 software Primer3input (http://bioinfo.ut.ee/primer3-0.4.0/, accessed on 12 January 2022).Primer sequences are provided in Supplementary Table S1.Amplification bands of pri-miRNA and pre-miRNAs are illustrated in Supplementary Figure S1E, demonstrating a single transcript for pri-miRNA miR-23a-miR-27a_miR-24-2 and specific amplifications for each pre-miRNA, i.e., pre-miRNA-23a, pre-miR-27a and pre-miR-24-2, respectively.No amplifications were observed in PCR control reactions containing only water as a template.Each PCR reaction was performed at least three times to obtain representative averages.The Livak method was used to analyze the relative quantification RT-qPCR data [106] and normalized in all cases, taking 100% as the wild-type (control) value, using Gapdh and Gusb as internal control for mRNA expression analyses, as previously described [103][104][105].

qRT-PCR Analyses (microRNA)
For microRNA expression analyses, 20 ng of total RNA was used for retrotranscription with a Universal cDNA Synthesis Kit II (Exiqon, Venlo, The Netherlands), and the resulting cDNA was diluted 1/80, following the manufacturer's guidelines.Real-time PCR experiments were performed with 1 µL of cDNA, GoTaq qPCR Master Mix (Promega) and corresponding primer sets, as described in Supplementary Table S1.All RT-qPCRs were performed using a CFX384TM thermocycler (Bio-Rad, Hercules, CA, USA) following the manufacturer's recommendations.The relative level of expression of each gene was calculated as described by Livak and Schmittgen [106] using 5S as an internal control for microRNA expression analyses.Each PCR reaction was performed at least three times to obtain representative averages.

Western Blot
Western blot was performed using 30 µg of total protein.The primary antibodies Mef2c (sc-13268; Santa Cruz Biotechnology, Dallas, TX, USA) and Tubulin (sc-8035; Santa Cruz Biotechnology, Dallas, TX, USA) were used at a concentration of 1:100 and 1:5000, respectively, and incubated overnight at 4 • C and the secondary antibody-HRP conjugate (#170-6516, Biorad, Hercules, CA, USA) at 1/5000 for 2 h at room temperature.Blocking was carried out with 5% milk and washed with PBST according to the antibody manufacturer's recommendations.

Luciferase Assay
Promotor distal sequence (L) was amplified from mouse genomic DNA with specific primers bearing HindIII/BamHI restriction sites and cloned into a pGLuc-Basic vector (New England Biolabs, Ipswich, MA, USA).3T3 fibroblasts (ATCC, Manassas, VA, USA) were co-transfected with 100 ng of the L-pGluc vector, 300 ng of pcLux vector control for internal normalization and 400 ng from Mef2c FL, Mef2c 3 ′ or Mef2c 5 ′ , respectively.Luciferase activity was measured 24 h after transfection by using the Pierce Gaussia Luciferase Flash Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) and normalized to pcLux vector control by using the Pierce Cypridina Luciferase Flash Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA).In all assays, transfections were carried out in triplicate.

Statistical Analyses
For statistical analyses of datasets, unpaired Student's t-tests were used.Significance levels or p values are stated in each corresponding figure legend.p < 0.05 was considered statistically significant.

Supplementary Materials:
The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/ncrna10030032/s1.Supplementary Figure S1.Panel (A).Western blot analysis demonstrating Mef2c pulldown assay as compared to IgG-negative control.Panel (B).Schematic representation of the MEF2C constructs, i.e., MEF2C FL, MEF2C 3 ′ del and MEF2C 5 ′ del.Note that within the MEF2C 3 ′ del construct, the sequences from a Pst1 site to the 3 ′ of the MEF2C gene are deleted, preserving the Srf, MADS-MEF2 and HJURP_C domains.On the other hand, within the MEF2C 5 ′ del construct, the sequences from the 5 ′ of the MEF2C gene to the ScaI site are deleted, therefore eliminating the Srf, MADS-MEF2 and HJURP_C domains.Panel (C).RT-qPCR analyses of Mef2c expression in HL1 atrial cardiomyocytes after transfection with the distinct MEF2C constructs and MEF2C siRNA.Observe that similar expression levels are obtained after transfection with MEF2C FL, MEF2C 5 ′ del and MEF2C 3 ′ del while MEF2C siRNA significantly diminished its expression.Panel (D).MEF2C protein expression levels in HL1 atrial cardiomyocytes after transfection with the distinct MEF2C constructs and MEF2C siRNA as revealed by Western blot.Panel (E).Transactivation analyses of the L fragment of the miR-23a-miR-27a-miR-24-2 locus with MEF2C FL, MEF2C 5 ′ del and MEF2C 3 ′ constructs.Observe that MEF2C FL and MEF2C 3 ′ constructs can transactivate this fragment while MEF2C 5 ′ del cannot transactivate it, as reported by luciferase assays.Supplementary Figure S2.Panel (A).RT-PCR analyses of pri-miRNA and pre-miRNA amplification products as revealed in gel electrophoresis.pre-miR-23a, pre-miR-27a and pre-miR-24-2 amplification resulted in products below 100 bp, as expected, while pri-miRNA amplification resulted in an approximately 350 bp band, as expected.Panel (B).Sanger sequencing of the pre-miR-23a, pre-miR-27a and pre-miR-24-2 amplicons and their corresponding analyses of sequence homology (blast).Supplementary Figure S3.Schematic representation of the miR-23a-miR-27a-mir-24-2 locus with its genomic sequence, upon which the pre-miRNA precursor sequences are highlighted (pre-miR-23a in yellow, pre-miR-27a in green and pre-miR-24-2 in pink).Primer sequences for pri-miRNA amplification are highlighted in blue, spanning from pre-miR-23a to pre-miR-24-2 sequence.Primers for pre-miRNA amplification are underlined.Supplementary Table S1.List of primer and siRNA sequences.

Figure 1 .
Figure 1.Panel (A) RT-qPCR analyses of the nuclear and cytoplasmic distribution of miR-23a_3p, miR-27a_3p and miR-24_3p mature microRNAs in HL1 cardiomyocytes.Note that all three mi-croRNAs are similarly expressed in the nucleus and cytoplasm in contrast to miR-130a, which is primarily cytoplasmic, and the long non-coding RNA Xist2, which is preferentially nuclear.Panel (B) RT-qPCR analyses of Mef2c pulldown assays for pre-miR-23a, pre-miR-27a and pre-miR-24, respectively.Note that increased levels are observed for pre-miR-23a and pre-miR-27a but not for pre-miR-24.Panel (C) RT-qPCR analyses of Mef2c pulldown assays for mature miR-23a_3p, miR-27a_3p and miR-24_3p, respectively.Note that none of the mature microRNAs are increased after Mef2c pulldown assays.Panel (D) RT-qPCR analyses of Mef2c pulldown assays for pri-miR-23-miR-27a-miR-24-2.Panel (E) Schematic representation of the Mef2c association with the miR-23a-miR-27a- Non-Coding RNA 2024, 10, x FOR PEER REVIEW 7 of 22

Figure 3 .
Figure 3. Panel (A) RT-qPCR analyses of distinct RNPs (Adar1, Ddx5, Ddx17, HnRNPa1, HnRNPa2b1, HnRNPa3 and Ksrp) in three distinct cell lines: 3T3 fibroblasts, HL1 cardiomyocytes and Sol8 skeletal muscle myoblasts.Observe that these RNPs display distinct expression levels on each of the tested cell lines.Panel (B) RT-qPCR analyses of the nuclear and cytoplasmic distribution of these RNPs in HL1 cardiomyocytes.Note that Adar1 is preferentially expressed in the nucleus, while Ddx5, Ddx17 and Ksrp are preferentially expressed in the cytoplasm.Panel (C) RT-qPCR analyses of RNP expression after over-expression of Mef2c full-length (FL), Mef2c 5′del, Mef2c 3′del and Mef2c siRNA in HL1 cardiomyocytes, respectively.Note that these RNPs are distinctly regulated by each of the Mef2c constructs analyzed.Panel (D) Schematic representation of the Mef2c 5′del and Mef2c 3′del regulation of the RNPs.All data are normalized to Gapdh expression.* p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3 .
Figure 3. Panel (A) RT-qPCR analyses of distinct RNPs (Adar1, Ddx5, Ddx17, HnRNPa1, HnRNPa2b1, HnRNPa3 and Ksrp) in three distinct cell lines: 3T3 fibroblasts, HL1 cardiomyocytes and Sol8 skeletal muscle myoblasts.Observe that these RNPs display distinct expression levels on each of the tested cell lines.Panel (B) RT-qPCR analyses of the nuclear and cytoplasmic distribution of these RNPs in HL1 cardiomyocytes.Note that Adar1 is preferentially expressed in the nucleus, while Ddx5, Ddx17 and Ksrp are preferentially expressed in the cytoplasm.Panel (C) RT-qPCR analyses of RNP expression after over-expression of Mef2c full-length (FL), Mef2c 5 ′ del, Mef2c 3 ′ del and Mef2c siRNA in HL1 cardiomyocytes, respectively.Note that these RNPs are distinctly regulated by each of the Mef2c constructs analyzed.Panel (D) Schematic representation of the Mef2c 5 ′ del and Mef2c 3 ′ del regulation of the RNPs.All data are normalized to Gapdh expression.* p < 0.05, ** p < 0.01, *** p < 0.001.
Non-Coding RNA 2024, 10, x FOR PEER REVIEW 14 of 22 deserves further analysis and might provide novel insights into the precise molecular mechanisms controlling the differential expression of the mature microRNAs of genomic clustered microRNAs.

4 .
Materials and Methods 4.1.MEF2C Pulldown Assays For the immunoprecipitation of endogenous MEF2C, protein A-Sepharose beads (Abcam, Cambridge, UK) were coated with 15 µg of antibody that recognized MEF2C (#9792-Cell Signalling) or control IgG (Abcam, Cambridge, UK) overnight at 4 • C with rotation.The next day, HL1 cells were lysed with PEB buffer (100 mM KCl, 5 mM MgCl 2, 10 mM Hepes, pH 7.0, 0.5% Nonidet P-40, 1 mM DTT, 100 units/mL RiboLOCK and Complete Protease Inhibitor Cocktail) for 10 min on ice and centrifuged at 10,000× g for 30 min at 4 • C. The supernatants were incubated with previously mentioned protein A-Sepharose-coated beads with 15 µg of antibody that recognized MEF2C or control IgG for 2 h at 4 • C with rotation, respectively.The corresponding beads were washed with NT2 buffer (50 mM Tris-HCl [Ph 7.5], 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40) two • C for 10 min, followed by 40 cycles of 95 • C for 30 s, 60 • C for 30 s and 72 • C for 30 s, with a final elongation step of 72