Alternatively spliced MBNL1 isoforms exhibit differential influence on enhancing brown adipogenesis

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

Contents lists available at ScienceDirect

BBA - Gene Regulatory Mechanisms journal homepage: www.elsevier.com/locate/bbagrm

Alternatively spliced MBNL1 isoforms exhibit differential influence on enhancing brown adipogenesis Ching-Sheng Hunga,b, Jung-Chun Lina,c,

⁎

a

PhD Program in Medicine Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan Department of Laboratory Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan c School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Alternative splicing Beige adipocyte MBNL1 RBM4a

Browning of white adipocytes (WAs) (also referred as beige cells) was demonstrated to execute thermogenesis by consuming stored lipids as do brown adipocytes (BAs), and this is highly related to metabolic homeostasis. Alternative splicing (AS) constitutes a pivotal mechanism for defining cellular fates and functional specifications. Nevertheless, the impacts of AS regulation on the browning of WAs have not been comprehensively investigated. In this study, we first identified the discriminative expression and splicing profiles of the muscleblind-like 1 (MBNL1) gene in postnatal brown adipose tissues (BATs) compared to those of embryonic BATs. A shift in the MBNL1+ex 5 isoform 7 (MBNL17) to MBNL1−ex 5 isoform 1 (MBNL11) was characterized throughout BAT development or during the in vitro browning of pre-WAs, 3T3-L1 cells. The interplay between MBNL1 and the exonic CCUG motif constitutes an autoregulatory mechanism for excluding MBNL1 exon 5. The simultaneous association of RNA-binding motif protein 4a (RBM4a) with exonic and intronic CU elements collaboratively mediates the skipping of MBNL1 exon 5. Overexpressing the MBNL11 isoform exhibited a more-prominent effect than that of the MBNL17 isoform on programming its own transcripts and beige cell-related splicing events in a CCUG motif-mediated manner. In addition to splicing regulation, overexpression of the MBNL11 and MBNL17 isoforms differentially enhanced beige adipogenic signatures of 3T3-L1 cells. Our findings demonstrated that MBNL1 constitutes an emerging and autoregulatory mechanism involved in development of beige cells.

1. Introduction Brown adipose tissues (BATs) composed of classical brown adipocytes (BAs) or browning white adipocytes (WAs) (also referred as beige adipocytes) execute non-shivering thermogenesis by [expending/consuming?] lipids to maintain the body temperature in infants and small rodents [1]. Classical BAs and myoblasts were demonstrated to originate from mesodermal progenitors expressing myogenic factor 5 (Myf5), the well-known myogenic transcription factor [2]. Cold exposure, treatment with β3-adrenergic receptor (β3-AR) agonists, and the presence of phytochemicals drive trans-differentiation of WAs to inducible beige cells, which was proposed as a therapeutic intervention to counteract obesity [3–5]. Differentiation of pre-BAs occurs with expression of the PR domain containing protein 16 (PRDM16) which drives increases in BA-selective factors to subsequently mediate the trans-differentiation of WAs [6,7]. PRDM16 triggers the transcription of peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ)

coactivator (PGC)-1α, a critical factor involved in BA-specific thermogenesis [8]. The PRDM16-mediated increase in the PPARα protein level promotes transcriptional activity of the PGC-1α promoter [9]. Nevertheless, the molecular mechanisms, including transcriptional and posttranscriptional regulation, which coordinate the concerted program required for the development of brown or beige adipogenesis are still largely uncharacterized. Alternative splicing (AS) functions as a critical mechanism for expanding genetic diversity to meet physiological functions of particular cell populations [10,11]. Modulation of RNA-binding proteins (RBPs), including serine/arginine-rich splicing factors (SRSFs), RNA-binding motif (RBM) proteins, muscleblind-like (MBNL) proteins, and CUG-BP and ETR3-like factors (CELFs), was extensively demonstrated to interfere with the development or maintenance of skeletal muscles which are derived from the same lineage as BAs [11–14]. Distinct RBPs were documented to coordinately reprogram AS networks which specify the development or functions of particular tissues or cell types [11–15]. For

⁎ Corresponding author at: School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 11031, Taiwan. E-mail address: [email protected] (J.-C. Lin).

https://doi.org/10.1016/j.bbagrm.2019.194437 Received 6 June 2019; Received in revised form 23 September 2019; Accepted 25 September 2019 1874-9399/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Ching-Sheng Hung and Jung-Chun Lin, BBA - Gene Regulatory Mechanisms, https://doi.org/10.1016/j.bbagrm.2019.194437

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen). To induce in vitro browning, 3T3-L1 cells were grown to confluence and then shifted to induction DMEM supplemented with 20% FBS, 0.5 mM IBMX (Invitrogen), 12.7 μM dexamethasone (Invitrogen), and 10 μg/ml insulin (Invitrogen) for 48 h. The differentiating DMEM (DM) supplemented with 10% FBS, 10 μg/ml insulin, 2 μM rosiglitazone, and 50 nM triiodothyronine was replaced with induction medium and replenished every 48 h for 6 days [19].

instance, micro (mi)RNA-mediated reduction of MBNL1 family members resulted in altered AS events which were relevant to the insufficient differentiation of a myogenic progenitor [16]. In addition to AS regulation, the alternative influence of MBNL1 of antagonizing the repressive effects of miRNAs on gene expression was related to the mortality and proliferation of embryonic stem cells [17]. Nevertheless, the potential impact of MBNL1 on classical BAs or beiges cells which are derived from distinct lineages through post-transcriptional regulation is still uncharacterized. In this study, the reprogrammed expressions and splicing profiles of MBNL1 transcripts were revealed during development of BATs or in vitro browning of pre-WAs, 3T3-L1 cells. A MBNL1-autoregulated or RNA-binding motif protein 4a (RBM4a)-mediated mechanism participated in programming splicing profiles of MBNL1 transcripts throughout beige adipogenesis. MBNL1 isoforms encoded from AS transcripts were demonstrated to exhibit differential impacts on beige adipogenesis-selective gene expressions, AS events, and beige adipogenic signatures.

2.5. Plasmid construction Open reading frames of mice MBNL1 isoforms 1 and 7 were amplified with a polymerase chain reaction (PCR) using a mouse fetal cDNA library as the template. The PCR product digested with Kpn I/ BamH I was inserted into the p3XFLAG-CMV14 vector (Sigma, St. Louis, MO, USA). The mouse genomic element containing the cassette exons of MBNL1, Mef2c, and FGFR2 was PCR-amplified using mice genomic DNA as the template. The PCR product was digested with Kpn I and BamH I restriction enzymes and inserted into the pcDNA 3.1 vector (Invitrogen). Derived mutants of the splicing reporters or RBM4a protein containing substituted nucleotides or amino acids were constructed using the QuikChange site-directed mutagenesis system (Stratagene, Amsterdam, The Netherlands). The vector-based short hairpin (sh)RNA targeting the mouse MBNL1 or RBM4a gene was purchased from the RNAi core facility at Academia Sinica (Taipei, Taiwan).

2. Materials and methods 2.1. Ethics statement for animal research RBM4a−/− C57BL/6 mice were generated according to a knockout design and genotype validation, details of which are provided in a previous report [18]. Animal care and following experiments were approved by the Institutional Animal Care and Use Committee of Taipei Medical University (No. LAC-2013-0208) and conducted according to relevant guidelines. All efforts were made to minimize animal suffering. After euthanization, interscapular brown and white fat tissues were collected from the same litter of wild-type (WT) and RBM4a−/− embryos or male WT and RBM4a−/− mice fed a regular diet for 8 weeks. Interscapular brown and white fat tissues were weighed and immediately frozen for RNA and protein extraction.

2.6. Transient transfection and reverse-transcription (RT)-PCR The plasmid was transfected using Lipofectamine 3000 according to the manufacturer's protocol (Invitrogen) into 3T3-L1 cells which were grown to 60%~70% confluence. To increase the transfection efficiency, transfection was repeated twice. Green fluorescent protein (GFP)transfected cells were applied to evaluate the transfection efficiency. 25%~30% transfection efficiency was consistently achieved using 3T3L1 cells throughout the study. Total RNA or cell lysates were extracted using a ReliaPrep RNA Miniprep kit (Promega, Madison, WI, USA). To minimize contamination by genomic DNA, extracted RNAs were treated with DNase I (Promega) prior to the following experiment. RNA (0.5 μg) was reverse-transcribed using SuperScriptase III (Invitrogen) in a 10-μL reaction and subjected to a PCR analysis using gene-specific primer sets (Supplementary Table 1). Signal densities of PCR-amplified transcripts were quantified using TotalLab Quant Software (Nonlinear, Durham NC, USA). A bar graph presents relative levels of alternatively spliced transcripts. The qPCR assay was conducted with SYBR green fluorescent dye and gene-specific primer sets (Supplementary Table 2) using an ABI One Step™ PCR machine (Applied Biosystems, Foster City, CA, USA). The relative messenger (m)RNA level was quantitated by the ΔΔ-Ct method, and normalized to the level of GAPDH mRNA. Quantitative results are shown as the mean ± standard deviation (SD). Statistical significance was determined using Student's unpaired t-test or an analysis of variance (ANOVA) test (* p < .05; ** p < .01; *** p < .005).

2.2. RNA extraction, complementary (c)DNA library construction, and sequencing Total RNAs were extracted using a PureLink RNA mini kit (Invitrogen, Camarillo, CA, USA) according to the manufacturer's protocol. The integrity of the prepared RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Redwood, CA, USA). Qualified total RNA (4 μg) was subjected to library construction using a NEBNext Ultra RNA Library Prep Kit from Illumina (NEB, Ipswich, MA, USA) according to the manufacturer's instructions. The constructed libraries were sequenced on the Illumina NextSeq 500 platform, and approximately 150-bp paired-end reads were generated. 2.3. Read mapping, transcript annotation, and quantification Preliminary reads were cleaned by trimming and removing poly-N or low-quality sequences (Q < 20). Filtered reads were aligned to the mouse reference genome (GRCm38) using the Tophat v2.0.9 program. Tolerance parameters were set to the default setting to allow mismatches of fewer than two bases. Aligned reads were used to generate transcriptome assemblies using the Cufflink program. Mutant loci within the assembled transcripts were identified using SAMtools. Transcriptome assemblies generated from individual samples were merged using the Cuffmerge utility to provide a standard for estimating transcript levels in each condition. Expression levels and statistical significance of the merged assemblies were calculated using Cuffdiff.

2.7. Immunoblot assay The extracted proteins were separated by using 10% polyacrylamide gel. An enhanced chemiluminescence system (Millipore, Billerica, MA, USA) was applied to perform the immunoblot analysis, and results were visualized using an LAS-4000 imaging system (Fujifilm, Tokyo, Japan). Primary antibodies used in this study included polyclonal anti-RBM4a (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc98,346), polyclonal anti-GAPDH (dilution 1:2000; MDBio, Taipei, Taiwan; 30,002), polyclonal anti-MBNL1 (Abcam, Cambridge, MA, USA; ab45899), and monoclonal anti-FLAG M2 (dilution 1:2000; Sigma; F1804). Signal intensities were evaluated using TotalLab Quant Software.

2.4. Cell culture and in vitro differentiation Mouse 3T3-L1 preadipocytes were cultured in growth medium (GM) composed of Dulbecco's modified Eagle medium (DMEM; Invitrogen, 2

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

prepared from BATs (Supplementary Table 3). MBNL17 was predominantly generated by embryonic BATs (Table 1B, FPKM = 1.006734) compared to that of MBNL11 (FPKM = 0.352765), whereas a more-significant increase in the MBNL11 transcript (log2 fold change = 3.208) than that of the MBNL17 transcript (log2 fold change = 1.522) was identified in postnatal BATs. As shown in Fig. 1A, reviewed sequences of seven alternatively spliced transcripts transcribed from the mouse MBNL1 gene were submitted to the National Center for Biotechnology Information (NCBI). The utilization of MBNL1 exons 1, 5, 7, and 8 was examined using RTPCR analyses to validate the results of whole transcriptome analyses. In addition to a gradual shift in the skipping of exon 5 throughout BAT development (Fig. 1B, upper, lanes 1–3), a gradual increase in inclusion of exons 7 and 8 (Supplementary Fig. 1A, upper panel) and the constitutive inclusion of exon 1 (Supplementary Fig. 1A, lower panel) indicated the relative levels of MBNL11 and MBNL17 transcripts during the BAT development. An increase in total MBNL1 transcripts was consistently validated using RT-PCR assays (Fig. 1B, middle, lanes 1–3) and qPCR analyses (Supplementary Fig. 2A) throughout BAT development. In vitro differentiation assays using 3T3-L1 cells which were extensively applied to study the development of WAs and beige cells was next conducted to verify the reprogrammed expression or splicing profiles of the MBNL1 transcript [21–23]. Upregulated expression of the PR domain containing 16 (PRDM16) transcript committed in vitro-cultured 3T3-L1 cells to beige adipogenesis (Fig. 1C, lower) [24]. Surprisingly, undulating splicing profiles of the MBNL1 transcripts were noted throughout the differentiation process of 3T3-L1 cells (Fig. 1C, upper). Nevertheless, the predominant expressions of MBNL11 and MBNL17 transcripts during in vitro differentiation of 3T3-L1 cells were consistently observed by validating the selection of MBNL1 exons 1, 7, and 8 (Supplementary Fig. 1B).Fluctuations in total MBNL1 transcripts were identified by RT-PCR assays (Fig. 1C, middle, lanes 1–6) and qPCR analyses (Supplementary Fig. 2B). Undulating levels of MBNL1 variants which reflected expression of the MBNL1 transcript were validated during in vitro browning of 3T3-L1 cells (Fig. 1C, right panel, lanes 1–3). The skipping of exon 5 (Fig. 1D, lane 1) or inclusion of exons 7 and 8 (Supplementary Fig. 1C, lane 1) consistently indicated relatively high levels of the MBNL11 transcript in mature BATs compared to those of mature WATs (Fig. 1D, lane 2; Supplementary Fig. 1C, lane 2) according to RT-PCR analyses. The relatively high expression of the total MBNL1 transcript in mature BATs compared to that of WATs was validated using RT-PCR (Fig. 1D, middle panel) and qPCR assays (Supplementary Fig. 2C). These results suggested the potential correlation between modulated MBNL1 transcripts expression and the browning of WAs.

2.8. Mitochondrial respiration assay The oxygen consumption rate (OCR; as an indicator of mitochondrial respiration) of in vitro-cultured cells was measured using a Seahorse XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA). In brief, 2 × 104 3T3-L1 cells were seeded in each well of Seahorse XF24 plates with 250 μL of DMEM and incubated overnight. Seeded cells were washed and immersed in 675 μL of unbuffered medium without CO2 for 1 h prior to the measurement. The basal and maximal OCRs, spare respiratory capacity, and ATP production were recorded following injection of complex-specific substrates including FCCP (2 μM), rotenone (2 μM), and oligomycin (2.5 μg/ml) in 8-min cycles as recommended. 2.9. Mitochondrial analysis In vitro cultured 3T3-L1 cells were shifted to DMEM containing100 nM MitoTracker Red FM (Invitrogen) for 30 min at 37 °C. Cells were washed with prewarmed culture medium and visualized using an Olympus IX81 microscope (Tokyo, Japan). The signal strength of captured pictures was analyzed using TotalLab Quant. 2.10. Oil-red-O staining In vitro-cultured 3T3-L1 cells were washed twice with phosphatebuffered saline (PBS) and fixed with 10% paraformaldehyde for 10 min at room temperature. Fixed cells were washed with PBS and rinsed with 60% isopropanol for 5 min at room temperature. Equilibrated cells were stained with a 0.3% filtered oil-red-O solution (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Stained cells were washed with distilled water three times. 2.11. RNA-protein interactions To investigate associations between proteins and transcripts, a FLAG-tagged protein expression vector was transfected into 3T3-L1 cells. At 24 h post-transfection, cell lysates were prepared using RIPA buffer containing 1% Nonidet P-40, followed by immunoprecipitation using anti-FLAG M2 beads (Sigma) at 4 °C for 2 h. The beads were washed with RIPA buffer containing 0.5% Nonidet P-40. Immunoprecipitated RNA was extracted using phenol/chloroform/isoamyl alcohol precipitation after proteinase K treatment and subjected to an RT-PCR analysis. 2.12. Statistical analyses An analysis of variance (ANOVA) and Student's t-tests were performed to determine the significance of the experimental results. p < .05 was considered statistically significant.

3.2. MBNL1 isoforms differentially autoregulate the skipping of MBNL1 exon 5 Binding of the MBNL1 protein to the CCTG site was previously reported to regulate distinct post-transcriptional events [25,26]. As shown in Fig. 2A, one CCTG site adjacent to the 5′ splice site of MBNL1 intron 5 was annotated (Fig. 2A, underlined characters). The splicing reporter containing cassette exons of MBNL1 and derived mutants were applied to demonstrate the impacts of distinct MBNL1 isoforms on utilization of MBNL1 exon 5 (Fig. 2A, lower). Using an in vitro differentiation assay, an increase in relative levels of the MBNL1−ex5 transcript generated from the WT MBNL1 reporter was identified (Fig. 2B, lanes 1–3), similar to that of the endogenous MBNL1 gene (Fig. 1B, upper). A thymine-to-adenosine substitution within the CCTG motif potentially strengthened the splicing efficiency of the downstream 5′ splice site, which was related to the generation of the MBNL1+ex5 transcript transcribed from the mutant reporter (Mut I) in both nondifferentiating and differentiating cells (Fig. 2C, lanes 3 and 4) compared to those of the WT MBNL1 reporter (Fig. 2C, lanes 1 and 2). Splicing profiles of the derived MBNL1 reporter (Mut II) containing the

3. Results 3.1. Expressions and splicing profiles of MBNL1 transcripts are reprogrammed throughout BAT development To investigate the impacts of post-transcriptional regulation on brown adipogenesis, whole-transcriptome analyses were conducted with total RNA samples prepared from embryonic (E13.5) and postnatal (P0) BATs [20]. Among the identified candidates, an increase in total MBNL1 transcripts was characterized in postnatal BATs (Table 1A, fragments per kilobase of transcript per million mapped reads (FPKM) = 6.2236) compared to that of embryonic BATs (Table 1A, FPKM = 1.4809) (n = 4). Splicing profiles of MBNL1 transcripts were also reprogrammed during brown adipogenesis. MBNL1 transcript 1 (MBNL11; NM_020007) and MBNL1 transcript 7 (MBNL17; NM_001310514) were predominantly identified in RNA samples 3

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

Table 1 Expression and splicing profiles of MBNL1 transcripts are reprogrammed throughout the development of brown adipose tissues (BATs). Whole-transcriptome analyses were conducted with total RNAs prepared from four pairs of independent tissues (n = 4) and analyzed using the CLC genomics workbench (GWB). (A) Expression levels and (B) splicing profiles of MBNL1 transcripts were identified in embryonic (E13.5) and postnatal (P0) BATs. A Gene

Locus

sample_1

sample_2

FPKM_1

FPKM_2

log2(fold_change)

p_value

q_value

MBNL1

chr3:60472830-60629750

BAT(E13.5)

BAT(P0)

1.4809

6.2236

2.071275

2.03E-04

0.015534

B Sample

Gene_name

Gene_id

Locus

Isoform

FPKM

TPM

log2(fold change)

p_value

q_value

BAT(E13.5) BAT(E13.5) BAT(P0) BAT(P0)

MBNL1 MBNL1 MBNL1 MBNL1

NM_001310514 NM_020007 NM_001310514 NM_020007

chr3:60472830-60629750 chr3:60472830-60629750 chr3:60472830-60629750 chr3:60472830-60629750

7 1 7 1

1.006734 0.352765 2.892474 3.259426

2.307497 1.01078 3.823394 4.664762

– – 1.522 3.208

1.55E-04 2.12E-04 1.94E-04 2.55E-04

0.024591 0.033012 0.015667 0.020144

Fig. 1. Splicing profiles of MBNL1 transcripts are reprogrammed throughout brown adipogenesis. (A) Diagram presents the exonic composition of alternatively spliced MBNL1 transcripts and the respective primer sets for RT-PCR assays. (B) Total RNAs and cell lysate prepared from four pairs of embryonic (E13.5 and E15.5) and postnatal (P0) brown adipose tissues (n = 4) were subjected to RT-PCR and immunoblotting assays using specific primers complementary to MBNL1 exons 4 and 6 and indicated antibodies. (C) Total RNAs isolated from non-differentiating (day 0) and differentiating cells (days 1–5) in four discrete experiments (n = 4) were subjected to RT-PCR assays using specific primer sets listed in a Supplementary Table 1. (D) Total RNAs prepared from four pairs of brown adipose tissues and white adipose tissues dissected from adult mice (8-week, n = 4) were subjected to RT-PCR assays using primer sets as previously described. Signal densities of the RT-PCR results were quantified using TotalLab Quant Software. The bar graph presents relative levels of the MBNL11 transcript. Quantitative results are shown as the mean ± SD. Statistical significance was determined using Student's unpaired t-test (* p < .05; ** p < .01; *** p < .005). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Fig. 2D, lane 3, relative level of MBNL1−ex5 = 63.14%) on enhancing generation of the MBNL1−ex5 transcript transcribed by the WT reporter. A thymine-to-adenosine substitution within the CCTG motif led to predominant expression of the MBNL1+ex5 transcript transcribed from the Mut I reporter in empty vector-transfectants or with overexpression of the MBNL17 isoform (Fig. 2D, lanes 4 and 6, relative levels of MBNL1−ex5 = 6.27% and 8.21%), whereas the presence of overexpression of the MBNL11 isoform resulted in an increase in the relative level of the MBNL1−ex5 transcript (Fig. 2D, lane 5, relative level of MBNL1−ex5 = 12.33%). In contrast, the mutation of CCTG to GCTG diminished the influence of overexpression of the MBNL1 isoform of enhancing levels of the MBNL1−ex5 transcript generated by the Mut II

mutation of CCTG to GCTG showed no response to the differentiating condition compared to that of non-differentiating cells (Fig. 2C, lanes 5 and 6), whereas expression of the MBNL1- ex5 transcript transcribed from the Mut II reporter was not abrogated in non-differentiating cells (Fig. 2C, lane 5) compared to that of the WT MBNL1 reporter (Fig. 2C, lane 1). The presence of an artificial CCUG site close to the authentic CCUG motif exhibited no influence on altering splicing profiles of the Mut III reporter (Fig. 2C, lanes 7 and 8) compared to that of the WT reporter in a similar condition (Fig. 2C, lanes 1 and 2). Overexpression of MBNL1 isoform 1 (MBNL11) encoded from the MBNL11 transcript exerted a more-prominent effect (Fig. 2D, lane 2, relative level of MBNL1−ex5 = 72.24%) than did the MBNL17 isoform 4

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

Fig. 2. Overexpression of MBNL1 enhances exclusion of MBNL1 exon 5 in a exonic motif-dependent manner. (A) Scheme shows the CCUG motif (underlined) within MBNL1 exon 5 (uppercase). The diagram presents the exonic composition of wild-type (WT) and mutant MBNL1 splicing reporters. (B) The WT MBNL1 splicing reporter was transfected into 3T3-L1 cells, followed by culturing in growth (day 0) or differentiating medium (days 1 and 2). (C) The WT or mutant MBNL1 splicing reporter was respectively transfected into 3T3-L1 cells, followed by culturing in growth (day 0) or differentiating medium (day 1). (D) The empty vector and expressing vector encoding the MBNL11 or MBNL17 isoform were respectively co-transfected with the WT or mutant MBNL1 splicing reporter into 3T3-L1 cells. Total RNAs and cell lysates extracted from four discrete experiments (n = 4) were subjected to an RT-PCR and immunoblotting assay with specific primer sets as listed in Supplementary Table 1 and indicated antibodies. Signal densities of the RT-PCR results were quantified using TotalLab Quant Software. The bar graph presents relative levels of the MBNL1−ex 5 transcript. Quantitative results are shown as the mean ± SD. Statistical significance was determined using Student's unpaired t-test (* p < .05; ** p < .01; *** p < .005).

reporter (Fig. 2D, lanes 8 and 9, relative levels of MBNL1−ex5 = 42.17% and 40.21%) compared to that of empty vector (EV)-transfectant (Fig. 2D, lane 7, relative level of MBNL1−ex5 = 32.74%). Taken together, the interplay between MBNL1 and the CCTG motif potentially lessened the strength of the downstream 5′ splice site, subsequently leading to the further exclusion of MBNL1 exon 5.

3.3. RBM4a functions as a splicing silencer toward selection of MBNL1 exon 5 RBM4a-regulated splicing events are relevant to the development and physiological signatures of BATs [20]. The simultaneous association of RBM4a with exonic and intronic CU elements frequently results in exclusion of alternatively spliced exons [27]. To validate potential impacts of RBM4a on utilization of MBNL1 exon 5, splicing profiles of MBNL1 transcripts were identified throughout the development of

5

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

Table 2 Depletion of RBM4a alters the expression and splicing profiles of MBNL1 transcripts in brown adipose tissues (BATs). Whole-transcriptome analyses were conducted with total RNAs prepared from four pairs of independent tissues (n = 4) and analyzed using the CLC genomics workbench (GWB). (A) Expression levels and (B) splicing profiles of MBNL1 transcripts were identified in postnatal (P0) BATs dissected from wild-type (WT) and RBM4a−/− mice. A Gene MBNL1

Locus

Sample_1

chr3:60472830-60629750

BAT(WT)

Sample_2 BAT(RBM4a

−/−

)

FPKM_1

FPKM_2

log2(fold_change)

p_value

q_value

6.2236

3.383416

−0.87927

1.57E-02

0.018221

B Sample

Gene_name

Gene_id

Locus

Isoform

FPKM

TPM

log2(fold change)

p_value

q_value

BAT(WT) BAT(WT) BAT(RBM4a−/−) BAT(RBM4a−/−)

MBNL1 MBNL1 MBNL1 MBNL1

NM_001310514 NM_020007 NM_001310514 NM_020007

chr3:60472830-60629750 chr3:60472830-60629750 chr3:60472830-60629750 chr3:60472830-60629750

7 1 7 1

2.892474 3.259426 2.224173 1.825623

3.823394 4.664762 2.909014 2.3878395

– – −0.37903 −0.83623

1.94E-04 2.55E-04 2.23E-04 2.17E-03

0.015667 0.020144 0.0142171 0.0212235

RBM4a−/− BATs. Results of whole-transcriptome analyses showed relatively low levels of total MBNL1 mRNA (Table 2A, FPKM = 3.383) and the MBNL11 transcript (Table 2B, FPKM = 1.8256) in adult RBM4a−/− BATs compared to those in WT BATs (Table 2A, FPKM = 6.224; Table 2B, FPKM = 3.259). As shown in Fig. 3A, the shift in exon 5-included MBNL17 to exon 5-skipped MBNL11 transcripts was diminished throughout the development of RBM4a−/− BATs (Fig. 3A, lanes 4–6) compared to those of the WT counterparts (Fig. 3A, lanes 1–3). Results of in vitro differentiation assays demonstrated that depletion of endogenous RBM4a resulted in relatively low levels of the MBNL11 transcript in both non-differentiating and differentiating 3T3L1 cells (Fig. 3B, lanes 3 and 4) compared to that of EV transfectants (Fig. 3B, lanes 1 and 2). A decrease in relative levels of the MBNL11 transcript generated by RBM4a−/− BATs or WATs was noted as well (Fig. 3C, lanes 2 and 4) compared to those of the WT counterparts (Fig. 3C, lanes 1 and 3). Transient overexpression of FLAG-tagged RBM4a (Fig. 3D, lane 2), but not SRSFs (Fig. 3D, lanes 3 and 4) or PTBP1 (Fig. 3D, lane 5), mediated a shift in MBNL17 to MBNL11 transcripts in 3T3-L1 cells. Interestingly, the presence of overexpressing CELF1, which shares a binding tendency toward the CCUG site, exhibited no effect on programming the splicing profile of the endogenous MBNL1 gene (Fig. 3D, lane 6). Collectively, these results indicated the specificity of the RBM4a-mediated mechanism for skipping MBNL1 exon 5.

MBNL11 (Fig. 4B, upper) or exon 5-excluded transcripts generated by the WT MBNL1 splicing reporter (Fig. 4B, lower; MBNL1−ex 5) showed no response to the engineered RBM4a containing mutant RNA recognition motifs (RRMs; Y37A, F39A, Y113A, and F115A) (Fig. 4B, lanes 3 and 9) or the cytoplasmic phosphomimetic S309D mutant (Fig. 4B, lanes 5 and 11) [29]. In contrast, the derived zinc knuckle domain-mutant (mZn; C162A, C165A) (Fig. 4B, lanes 4 and 10) and the nuclear non-phosphorylatable S309A mutant (Fig. 4A, lanes 6 and 12) exhibited similar effects to that of WT RBM4a (WT) (Fig. 4A, lanes 2 and 8) on enhancing exclusion of MBNL1 exon 5. These results consistently verified the influence of RBM4a on modulating nuclear regulation, such as AS events, through its binding activity to pre-mRNAs as reported in our previous studies [20,29]. Results of the RNA-protein association analyses showed that the association of FLAG-tagged MBNL1 or RBM4a proteins with MBNL11 transcripts (Fig. 4C, lanes 5 and 6) was correlated to the shift in MBNL17 to MBNL11 transcripts (Fig. 4C, lanes 2 and 3) compared to EV-transfected cells (Fig. 4C, lane 1). This result further demonstrated the impaired effect of the engineered RBM4a protein containing mutant RRM or the cytoplasmic phosphomimetic residue on regulating AS events. Taken together, an additional mechanism through which RBM4a regulates exclusion of MBNL1 exon 5 was disclosed in this study.

3.5. MBNL1 isoforms differentially modulate BA-related splicing events In addition to autoregulation, potential impacts of MBNL1 isoforms on brown adipogenesis-related AS events [20,29] were next investigated. Among these candidates, including pyruvate kinase muscle (PKM), insulin receptor (IR), fibroblast growth factor receptor 2 (FGFR2), and Myocyte enhancer factor 2C (MEF2C), the CCUG motif was annotated within the alternatively spliced exon. The presence of the overexpressed MBNL11 isoform resulted in enhancements of relative levels of mature BA-specific PKM1, FGFR2 IIIb, INSR-B, and Mef2c+exβ transcripts (Fig. 5A, lane 2) compared to those of the EV transfectants (Fig. 5A, lane 1). The existence of the MBNL17 isoform exhibited a lessprominent effect than did MBNL11 on programming BA-related splicing profiles (Fig. 5A, lane 3). In contrast, shRNA-mediated knockdown of endogenous MBNL1 lessened the influence of the differentiating condition on driving the shift in embryonic PKM2, FGFR2 IIIc, INSR-A, and Mef2c−exβ transcripts (Fig. 5B, lanes 3 and 5) to BA-related transcripts in differentiating 3T3-L1 cells (Fig. 5B, lanes 4 and 6) compared to that of EV-transfected cells (Fig. 5B, lanes 1 and 2). Splicing reporters containing cassette exons of Mef2c, the FGFR2 gene, or derived mutants were established to further demonstrate impacts of the interplay between MBNL1 and the CCUG motif (Fig. 5C, left, underlined characters) on regulating AS events. An increase in the Mef2c+exβ transcript generated from the WT Mef2c reporter or the FGFR2+ex8a transcript

3.4. RBM4a regulates the selection of MBNL1 exon 5 in a CU elementdependent manner Simultaneous interplay of RBM4a with exonic and intronic CU elements adjacent to the 5′ splice site was demonstrated to frequently mediate exclusion of AS exons [27]. The sole and direct binding of RBM4a to the intronic CU element next to the 5′ splice site conversely enhanced inclusion of the upstream exon [28]. To further examine the influence of RBM4a on utilization of MBNL1 exon 5, spliced transcripts generated by the MBNL1 splicing reporter or derived mutant were identified in the presence of overexpression of the RBM4a protein. Overexpression of RBM4a drove the shift in MBNL1+ex5 to MBNL1−ex5 transcripts generated from the WT MBNL1 reporter (Fig. 4A, lane 2) compared to EV transfectants (Fig. 4A, lane 1). Disruption of the exonic CCTG site dominantly interfered with the influence of exogenous RBM4a on enhancing relative levels of the MBNL1−exon 5 transcript transcribed from the Mut I (Fig. 4A, lane 4) or Mut II reporter (Fig. 4A, lane 6) compared to the EV-transfectant (Fig. 4A, lanes 3 and 5). In contrast, overexpression of RBM4a resulted in elevation of the MBNL1+ex5 transcript generated from the mutant reporter containing the interrupted CU element (Fig. 4A, Mut III, lane 8) compared to that of the EV transfectant (Fig. 4A, lane 7). The abundance of endogenous 6

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

Fig. 3. RBM4a regulates the splicing profiles of MBNL1 transcripts throughout brown adipogenesis. (A) Total RNAs were extracted from four pairs of embryonic (E13.5 and E15.5) and postnatal (P0) brown adipose tissues (BATs) dissected from wild-type (WT) or RBM4a−/− mice. (B) The empty vector or targeting vector of RBM4a was respectively transfected into 3T3-L1 cells, followed by culturing in growth (day 0) or differentiating medium (day 1). (C) Total RNAs were prepared from four pairs of BATs or white adipose tissues (WATs) dissected from adult WT or RBM4a−/− mice (8 weeks old, n = 4). (D) The empty vector or expressing vector for encoding distinct splicing regulators was respectively transfected into 3T3-L1 cells, followed by culturing in growth medium. Total RNAs and cell extracts were isolated from four discrete experiments (n = 4) and subjected to RT-PCR and immunoblot analyses using specific primer sets and indicated antibodies. Signal densities of the RT-PCR results were quantified using TotalLab Quant Software. The bar graph presents relative levels of MBNL11 transcripts. Quantitative results are shown as the mean ± SD. Statistical significance was determined using Student's unpaired t-test (* p < .05; ** p < .01; *** p < .005). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

produced from the WT FGFR2 reporter was identified with overexpression of the MBNL11 isoform (Fig. 5C, right, lane 2) compared to that of EV transfectants (Fig. 5C, right, lane 1). Splicing profiles of mutant Mef2c or FGFR2 reporters (Mut I) containing substituted nucleotides within the proximal CCUG sequence remained unchanged in MBNL11-overexpressing cells and EV transfectants (Fig. 5C, right, lanes

3 and 4). Nevertheless, overexpression of the MBNL11 protein exerted similar effects of enhancing relative levels of the Mef2c+exβ or FGFR2+e 8a transcript generated from the engineered reporter (Mut II) containing substituted nucleotides within the distal CCUG sequence (Fig. 5C, right, lanes 5 and 6). Taken together, these results showed differential influences of the MBNL1 isoform on BA-related splicing events in a CCUG 7

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

Fig. 4. RBM4a induces exclusion of MBNL1 exon 5 through a sequence-dependent mechanism. (A) The empty vector and expressing vector encoding the RBM4a protein were respectively co-transfected with the wild-type (WT) or mutant MBNL1 splicing reporter into 3T3-L1 cells, followed by culturing in growth medium. (B) The empty vector, WT RBM4a-overexpressing vector, or engineered mutants were respectively transfected alone or co-transfected with the WT MBNL1 splicing reporter into 3T3-L1 cells. Total RNAs and cell lysates were extracted from four discrete experiments (n = 4) and subjected to RT-PCR and immunoblot assays using indicated primer sets and antibodies. (C) The empty vector and expressing vector encoding FLAG-tagged MBNL11 or RBM4a were respectively transfected into 3T3L1 cells in four discrete experiments (n = 4). Cell extracts were prepared and subjected to an immunoprecipitation experiment using an anti-FLAG antibody. Immunoprecipitated RNAs were extracted and subjected to RT-PCR assays using primer sets complementary to the MBNL1 transcript. Signal densities of the RT-PCR results were quantified using TotalLab Quant Software. The bar graph presents relative levels of the MBNL11 or MBNL1−ex 5transcript. Quantitative results are shown as the mean ± SD. Statistical significance was determined using Student's unpaired t-test (* p < .05; ** p < .01; *** p < .005).

cell and tissue development has increased with the refinement of highthroughput approaches and bioinformatics analyses [32,33]. In this study, autoregulation of MBNL1 splicing constitutes an emerging circuit which programs BA-associated splicing networks. The regulatory mechanisms through which splicing factors program splicing events are complex and are still being debated due to the diversity of responsive elements [34]. Sequestration of MBNL1 by expanded CUG/CCUG repeats within the 3′ untranslated region (UTR) of DMPK transcripts is considered a pathological cause of myotonic dystrophy [35,36]. The interplay between MBNL1 and the CUG element within cardiac troponin T (cTNT) intron 4 was demonstrated to compete with the binding of U2AF65 to the 3′ splice site, resulting in exclusion of downstream cTNT exon 5 [37]. In contrast, binding of MBNL1 to INSR intron 11 next to the 5′ splice site enhanced recruitment of U1 snRNP, resulting in upregulated levels of exon 11-included INSR-B transcripts [38]. MBNL1 functions as a splicing silencer toward exon 7A of chloride channel 1 (CLCN1) by binding to the exonic CCUG sequence [39]. The direct binding of the zinc finger motif of MBNL1 to the GC dinucleotide embedded within polypyrimidines was characterized using systematic evolution of ligands by exponential enrichment (SELEX) and in vitro mobility shift assay [40,41]. Results of crosslinking and immunoprecipitation sequencing demonstrated high-affinity binding of the MBNL1 protein to the CTGCT motif within MBNL1 exon 1 [42]. Nevertheless, yeast three-hybrid studies demonstrated that MBNL1 preferably binds to symmetric bulges of CHHG or CHG motif (H corresponds to A, C or U) [43,44]. Other reports also documented the binding tendency of MBNL1 to a CAG or CCG motif which is considered a thermodynamically stable structure in addition to CUG or YGCY sequence [45,46]. In addition to the cis responsive element, the impact of MBNL1 on alternative splicing regulation is manipulated by the coordinative splicing factors. MBNL1 and polypyrimidine tract binding protein 1 (PTBP1) were revealed to exhibit opposing influence on the utilization of striated muscle-related exon [47,48]. In contrast, MBNL and PTB cooperatively repressed the inclusion of α-tropomyosin exon 3 through direct interaction with respective element [49]. Taken these studies together, the interplay between MBNL1 and the corresponding cis motifs (YGCY or CCTG) or collaborative trans-factor may constitute a

sequence-dependent manner. 3.6. MBNL1 isoforms exert differential influences on brown adipogenic signatures An increase in the total MBNL1 transcript throughout in vivo BAT development (Fig. 1B) suggests its potential impacts on brown adipogenesis. To validate this inference, in vitro functional assays were conducted with overexpression of distinct MBNL1 isoform in 3T3-L1 cells. Overexpression of the FLAG-tagged MBNL11 isoform resulted in increased levels of the PRDM16, UCP1, and CITED1 transcripts (Fig. 6A, lane 2) encoding functional proteins which are essential to the development or function of mature BAs [24,30]. In contrast, overexpression of the MBNL17 isoform exhibited a more-minor influence than did MBNL11 on enhancing BA-related gene expressions (Fig. 6A, lane 3). More lipid droplets were visualized in MBNL11-overexpressing 3T3-L1 cells (Fig. 6B, MBNL11) compared to MBNL17-overexpressing cells (Fig. 6B, MBNL17) using oil-red-O staining. The existence of overexpressed MBNL1 enhanced the mitochondriogenesis of transfected cells compared to that of EV-transfected cells cultured in proliferating medium (Fig. 6C). Nevertheless, mitochondria were more abundant in cells overexpressing MBNL11 (Fig. 6C, MBNL11 and bar chart) than in MBNL17-overexpressing cells (Fig. 6C, MBNL17). Accordingly, MBNL11overexpressing cells exhibited relatively high basal, maximal, and spare capacities of the OCR and ATP production (Fig. 6D, red line and bar) compared to EV-transfected cells (Fig. 6D, blue line and bar) cultured in proliferating medium. The minor influence of MBNL17 overexpression on enhancing the activity of mitochondrial respiration compared to that of the MBNL11 isoform was consistently noted (Fig. 6D, green line and bar). These results indicated a discriminative effect of the MBNL1 isoform on brown adipogenic signatures. 4. Discussion The AS mechanism constitutes a pivotal mechanism for generating spatial isoforms to meet the requirements of a particular cell type or tissue, including BAs [31]. Interest in complex AS networks involved in 8

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

Fig. 5. Interplay between MBNL1 and the CCUG motif programs brown adipocyte (BA)-related splicing events. (A) The empty vector and expressing vector encoding the MBNL11 or MBNL17 isoform were respectively transfected into 3T3-L1 cells. (B) The empty vector and targeting vector of MBNL1 were respectively transfected into 3T3-L1 cells, followed by culturing in growth (day 0) or differentiating medium (day 1). (C) The scheme shows the CCUG motif (underlined) within Mef2c exon β and FGFR2 exons 8a/8b. The diagram presents the exonic composition of the wild-type (WT) or mutant splicing reporters of the Mef2c and FGFR2 genes. The empty vector or expressing vector encoding the MBNL11 isoform was respectively co-transfected with the WT or mutant splicing reporter into 3T3-L1 cells. Total RNAs and protein extracts were extracted from four discrete experiments (n = 4), followed by RT-PCR and immunoblot assays using indicated antibodies and specific primer sets as listed in Supplementary Table 1. Signal densities of the RT-PCR results were quantified using TotalLab Quant Software. The bar graph presents relative levels of the indicated transcripts. Quantitative results are shown as the mean ± SD. Statistical significance was determined using Student's unpaired t-test (* p < .05; ** p < .01; *** p < .005). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Autoregulated AS constitutes a common mechanism for fine-tuning expression profiles or biological activities of splicing regulators [50,51]. During striated myogenesis, an increase in the MBNL1 protein enhanced its binding to a YGCY site within MBNL1 exon 1, subsequently inducing generation of the MBNL1−ex 1 transcript which exhibited weaker translational activity than did the full MBNL1 transcript [52]. Depletion of MBNL1 was reported to mediate an increase in levels of the MBNL1−ex 7 transcript generated from the endogenous MBNL1 gene or MBNL1 splicing reporter, and this was considered another autoregulatory mechanism [53]. The interplay between MBNL1 and the YGCY motif close to the 3′ splice site of MBNL1 intron 4 was demonstrated to weaken the strength of the 3′ splice site of MBNL1 intron 4,

coordinated mechanism for manipulating the impact of MBNL1 on variable AS events. In this study, results of splicing reporter assays demonstrated that the CCTG motif functioned as an exonic splicing silencer (ESS) to the adjacent 5′ splice site, leading to the exclusion of exon 5 in most MBNL1 variants. The existence of upregulated MBNL1 further strengthened the activity of ESS, leading to the reduced levels of the exon 5-included MBNL11 transcript in particular stages or cell types, such as brown or beige adipogenesis. The opposite impact of overexpressed MBNL1 on facilitating expression of Mef2c+exβ and FGFR2+ex 8a transcripts via the exonic CTG element could be relevant to other splicing regulator or mechanism, which should be further demonstrated with more related experiment. 9

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

Fig. 6. MBNL1 isoforms exhibit differential impacts on enhancing brown adipogenesis. (A) The empty vector or expressing vector encoding the MBNL1 isoform was respectively transfected into 3T3-L1 cells. Expression profiles of brown adipocyte (BA)-related factors were analyzed in four discrete experiments (n = 4) using RTPCR and qPCR assays with specific primer sets as listed in Supplementary Tables 1 and 2. (B and C) The empty or expressing vector encoding the MBNL1 isoform was transfected into 3T3-L1 cells and subjected to oil-red-O staining in four discrete experiments (n = 4). (C) Parallel transfections were performed using 3T3-L1 cells. Mitochondrial abundance was validated by epifluorescence in living cells using the Mitotracker Red FM dye in four discrete experiments (n = 4). Fluorescence intensities of stained cells were quantified using TotalLab Quant Software. (D) 3T3-L1 cells were transfected with the empty vector or expressing vector encoding the MBNL1 isoform, followed by culturing in growth medium for 24 h, and then were subjected to bioenergetic analyses. The bar graph shows mean values of basal and maximal oxygen consumption rates, spare capacity, and ATP production in four discrete experiments using a Seahorse XF24 Bioanalyzer (n = 4). Results are presented as the mean ± SD, and statistical significance was determined with Student's unpaired t-test (* p < .05; ** p < .01; *** p < .005). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Collectively, utilization of MBNL1 exons 5 and 7 may constitute a coordinated mechanism for manipulating the cellular localization and regulatory activities of MBNL11 and MBNL17 isoforms in tissue- or disease-related AS events. In this study, the discriminative expressions and splicing profiles of MBNL1 isoforms were disclosed throughout brown and beige adipogenesis. Selection of MBNL1 exon 5 was regulated via autoregulation or coordinated regulation. Differential impacts of the MBNL11 and MBNL17 isoforms on beige adipogenesis-related gene expressions and AS events were demonstrated, which are relevant to the development and physiological signatures of beige cells.

which led to exclusion of MBNL1 exon 5 [54]. In this study, results of splicing reporter assays showed that the interplay between MBNL1 and the exonic CCUG site near the 5′ splice site resulted in exclusion of MBNL1 exon 5. In addition to MBNL1-mediated autoregulation, the interplay between RBM4a and the exonic CCUU motif was demonstrated to constitute an additional mechanism for repressing inclusion of MBNL1 exon 5. MBNL1 acted as an exonic splicing silencer or intronic splicing enhancer as did the RBM4a protein toward utilization of the AS exon. The simultaneous interplay of MBNL1 or RBM4a with exonic and intronic motifs may constitute a coordinated mechanism that participates in excluding regulated exons, including MBNL1 exon 5. These results implied that determining how MBNL1 programs AS event to meet the needs of beige cells and other cell types is still an enigma and requires further study. Cellular localization of the splicing regulator constitutes another mechanism for manipulating its influence on nuclear events, including AS regulation [55,56]. Two classes of nuclear localization signals (NLSs), the well-known bipartite NLS and conformational NLS, were defined within the MBNL1 protein [57]. Inclusion of MBNL1 exons 7 and 8 participates in the coding of the bipartite NLS within the MBNL1 protein. Nuclear and cytoplasmic distributions of the MBNL1−ex7 isoform were identified, whereas the majority of the MBNL1+ex7 isoform was deposited in the nuclear fraction [58]. Autoregulation of MBNL1 exon 7 therefore acted as a feedback loop to maintain the homeostasis of MBNL1 abundance and regulatory activity in nuclei [53,58]. An increase in the MBNL1+ex 7 isoform was reported to alter splicing events which were related to active metastasis or the antiapoptotic signature of in vitro-cultured cancer cells [58]. The presence of the MBNL1 exon 5-encoded region was also demonstrated to modulate nuclear localization of the MBNL1 protein [59]. In addition, the presence of the MBNL1 exon 7-encoded region was essential for homodimerization of MBNL1 isoforms, functioning as another mechanism for manipulating their cellular deposition and corresponding functions [58,60]. In addition to cellular localization or protein interaction, the presence of the exon 7-encoding region was documented to result in high affinity between MBNL1 variants and the corresponding CUG sequence [60].

Transparency document The Transparency document associated with this article can be found, in online version. Declaration of competing interest All authors declare that no conflicts of interest exist. Acknowledgments This work was supported by a grant (MOST107-2320-B-038-060) from the Ministry of Science and Technology, Taiwan. The authors declare that no competing interests exist. Author contributions Jung-Chun Lin designed and performed the experiments, analyzed the results, and wrote the manuscript. Ching-Sheng Hung performed the experiments. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// 10

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

doi.org/10.1016/j.bbagrm.2019.194437. [26]

References [1] X. Zeng, M. Ye, J.M. Resch, M.P. Jedrychowski, B. Hu, B.B. Lowell, D.D. Ginty, B.M. Spiegelman, Innervation of thermogenic adipose tissue via a calsyntenin 3βS100b axis, Nature 569 (2019) 229–235. [2] M. Oka, N. Kobayashi, K. Matsumura, M. Nishio, K. Saeki, Exogenous cytokine-free differentiation of human pluripotent stem cells into classical brown adipocytes, Cells 8 (2019) (pii: E373). [3] Y. Jiang, D.C. Berry, J.M. Graff, Distinct cellular and molecular mechanisms for β3 adrenergic receptor-induced beige adipocyte formation, Elife 6 (2017) e30329. [4] M. Okla, J. Kim, K. Koehler, S. Chung, Dietary factors promoting brown and beige fat development and thermogenesis, Adv. Nutr. 8 (2017) 473–483. [5] K. Velickovic, H.A. Lugo Leija, I. Bloor, J. Law, H. Sacks, M. Symonds, V. Sottile, Low temperature exposure induces browning of bone marrow stem cell derived adipocytes in vitro, Sci. Rep. 8 (2018) 4974. [6] I.J. Lodhi, J.M. Dean, A. He, H. Park, M. Tan, C. Feng, H. Song, F.F. Hsu, C.F. Semenkovich, PexRAP inhibits prdm16-mediated thermogenic gene expression, Cell Rep. 20 (2017) 2766–2774. [7] L. He, M. Tang, T. Xiao, H. Liu, W. Liu, G. Li, F. Zhang, Y. Xiao, Z. Zhou, F. Liu, F. Hu, Obesity-associated mir-199a/214 cluster inhibits adipose browning via prdm16-pgc-1α transcriptional network, Diabetes 67 (2018) 2585–2600. [8] P. Seale, S. Kajimura, B.M. Spiegelman, Transcriptional control of brown adipocyte development and physiological function–of mice and men, Genes Dev. 23 (2009) 788–797. [9] E. Hondares, M. Rosell, J. Díaz-Delfín, Y. Olmos, M. Monsalve, R. Iglesias, F. Villarroya, M. Giralt, Peroxisome proliferator-activated receptor α (PPARα) induces PPARγ coactivator 1α (PGC-1α) gene expression and contributes to thermogenic activation of brown fat: involvement of PRDM16, J. Biol. Chem. 286 (2011) 43112–43122. [10] Y. Song, O.B. Botvinnik, M.T. Lovci, B. Kakaradov, P. Liu, J.L. Xu, G.W. Yeo, Singlecell alternative splicing analysis with expedition reveals splicing dynamics during neuron differentiation, Mol. Cell 67 (2017) 148–161. [11] A. Bertero, P.A. Fields, V. Ramani, G. Bonora, G.G. Yardimci, H. Reinecke, L. Pabon, W.S. Noble, J. Shendure, C.E. Murry, Dynamics of genome reorganization during human cardiogenesis reveal an RBM20-dependent splicing factory, Nat. Commun. 10 (2019) 1538. [12] N. Xie, M. Chen, R. Dai, Y. Zhang, H. Zhao, Z. Song, L. Zhang, Z. Li, Y. Feng, H. Gao, L. Wang, T. Zhang, R.P. Xiao, J. Wu, C.M. Cao, SRSF1 promotes vascular smooth muscle cell proliferation through a Δ133p53/EGR1/KLF5 pathway, Nat. Commun. 8 (2017) 16016. [13] C.M. Chamberlain, L.P. Ranum, Mouse model of muscleblind-like 1 overexpression: skeletal muscle effects and therapeutic promise, Hum. Mol. Genet. 21 (2012) 4645–4654. [14] Y. Blech-Hermoni, C.B. Sullivan, M.W. Jenkins, O. Wessely, A.N. Ladd, CUG-BP, Elav-like family member 1 (CELF1) is required for normal myofibrillogenesis, morphogenesis, and contractile function in the embryonic heart, Dev. Dyn. 245 (2016) 854–873. [15] S.M. Weyn-Vanhentenryck, H. Feng, D. Ustianenko, R. Duffié, Q. Yan, M. Jacko, J.C. Martinez, M. Goodwin, X. Zhang, U. Hengst, S. Lomvardas, M.S. Swanson, C. Zhang, Precise temporal regulation of alternative splicing during neural development, Nat. Commun. 9 (2018) 2189. [16] B.W. Zhang, H.F. Cai, X.F. Wei, J.J. Sun, X.Y. Lan, C.Z. Lei, F.P. Lin, X.L. Qi, M. Plath, H. Chen, miR-30-5p regulates muscle differentiation and alternative splicing of muscle-related genes by targeting MBNL, Int J Mol Sci. 17 (2016) (pii: E182). [17] D.R. Wu, K.L. Gu, J.C. Yu, X. Fu, X.W. Wang, W.T. Guo, L.Q. Liao, H. Zhu, X.S. Zhang, J. Hui, Y. Wang, Opposing roles of miR-294 and MBNL1/2 in shaping the gene regulatory network of embryonic stem cells, EMBO Rep. 19 (2018) (pii: e45657). [18] J.C. Lin, Y.T. Yan, W.K. Hsieh, P.J. Peng, C.H. Su, W.Y. Tarn, RBM4 promotes pancreas cell differentiation and insulin expression, Mol. Cell. Biol. 33 (2013) 319–327. [19] H. Asano, Y. Kanamori, S. Higurashi, T. Nara, K. Kato, T. Matsui, M. Funaba, Induction of beige-like adipocytes in 3T3-L1 cells, J. Vet. Med. Sci. 76 (2014) 57–64. [20] J.C. Lin, Y.L. Chi, H.Y. Peng, Y.H. Lu, RBM4-Nova1-SRSF6 splicing cascade modulates the development of brown adipocytes, Biochim. Biophys. Acta 1859 (2016) 1368–1379. [21] W. Wang, M. Kissig, S. Rajakumari, L. Huang, H.W. Lim, K.J. Won, P. Seale, Ebf2 is a selective marker of brown and beige adipogenic precursor cells, Proc. Natl. Acad. Sci. U. S. A. 111 (2014) 14466–14471. [22] J. Lone, J.H. Choi, S.W. Kim, J.W. Yun, Curcumin induces brown fat-like phenotype in 3T3-L1 and primary white adipocytes, J. Nutr. Biochem. 27 (2016) 193–202. [23] Y. Unno, H. Yamamoto, S. Takatsuki, Y. Sato, T. Kuranaga, K. Yazawa, Y. Ono, T. Wakimoto, Palmitoyl lactic acid induces adipogenesis and a brown fat-like phenotype in 3T3-L1 preadipocytes, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1863 (2018) 772–782. [24] G. Alemán, A.L. Castro, A. Vigil-Martínez, I. Torre-Villalvazo, A. Díaz-Villaseñor, L.G. Noriega, I. Medina-Vera, G. Ordáz, N. Torres, A.R. Tovar, Interaction between the amount of dietary protein and the environmental temperature on the expression of browning markers in adipose tissue of rats, Genes Nutr. 14 (2019) 19. [25] E. deLorimier, M.N. Hinman, J. Copperman, K. Datta, M. Guenza, J.A. Berglund, Pseudouridine modification inhibits muscleblind-like 1 (MBNL1) binding to CCUG

[27] [28] [29] [30] [31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

11

repeats and minimally structured RNA through reduced RNA flexibility, J. Biol. Chem. 292 (2017) 4350–4357. C. Sellier, E. Cerro-Herreros, M. Blatter, F. Freyermuth, A. Gaucherot, F. Ruffenach, P. Sarkar, J. Puymirat, B. Udd, J.W. Day, G. Meola, G. Bassez, H. Fujimura, M.P. Takahashi, B. Schoser, D. Furling, R. Artero, F.H.T. Allain, B. Llamusi, N. Charlet-Berguerand, rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences, Nat. Commun. 9 (2018) 2009. J.C. Lin, W.Y. Tarn, RBM4 down-regulates PTB and antagonizes its activity in muscle cell-specific alternative splicing, J. Cell Biol. 193 (2011) 509–520. J.C. Lin, W.Y. Tarn, Exon selection in alpha-tropomyosin mRNA is regulated by the antagonistic action of RBM4 and PTB, Mol. Cell. Biol. 22 (2005) 10111–10121. J.C. Lin, RBM4-MEF2C network constitutes a feed-forward circuit that facilitates the differentiation of brown adipocytes, RNA Biol. 12 (2015) 208–220. N.H. Kang, S. Mukherjee, J.W. Yun, Trans-cinnamic acid stimulates white fat browning and activates brown adipocytes, Nutrients 11 (2019) (pii: E577). M. Aprile, S. Cataldi, M.R. Ambrosio, V. D'Esposito, K. Lim, A. Dietrich, M. Blüher, D.B. Savage, P. Formisano, A. Ciccodicola, V. Costa, PPARγΔ5, a naturally occurring dominant-negative splice isoform, impairs PPARγ function and adipocyte differentiation, Cell Rep. (2018) 1577–1592. C. Trapnell, B.A. Williams, G. Pertea, A. Mortazavi, G. Kwan, M.J. van Baren, S.L. Salzberg, B.J. Wold, L. Pachter, Transcript assembly and quantification by RNASeq reveals unannotated transcripts and isoform switching during cell differentiation, Nat. Biotechnol. 28 (2010) 511–515. Y. Zhang, K. Chen, S.A. Sloan, M.L. Bennett, A.R. Scholze, S. O'Keeffe, H.P. Phatnani, P. Guarnieri, C. Caneda, N. Ruderisch, S. Deng, S.A. Liddelow, C. Zhang, R. Daneman, T. Maniatis, B.A. Barres, J.Q. Wu, An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex, J. Neurosci. (2014) 11929–11947. Y. Xue, Y. Zhou, T. Wu, T. Zhu, X. Ji, Y.S. Kwon, C. Zhang, G. Yeo, D.L. Black, H. Sun, X.D. Fu, Y. Zhang, Genome-wide analysis of PTB-RNA interactions reveals a strategy used by the general splicing repressor to modulate exon inclusion or skipping, Mol. Cell 36 (2009) 996–1006. O.J. Pettersson, L. Aagaard, D. Andrejeva, R. Thomsen, T.G. Jensen, C.K. Damgaard, DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1, Nucleic Acids Res. 42 (2014) 7186–7200. R.B. Siboni, M. Nakamori, S.D. Wagner, A.J. Struck, L.A. Coonrod, S.A. Harriott, D.M. Cass, M.K. Tanner, J.A. Berglund, Actinomycin D specifically reduces expanded CUG repeat RNA in myotonic dystrophy models, Cell Rep. 13 (2015) 2386–2394. M.B. Warf, J.V. Diegel, P.H. von Hippel, J.A. Berglund, The protein factors MBNL1 and U2AF65 bind alternative RNA structures to regulate splicing, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 9203–9208. S. Sen, I. Talukdar, Y. Liu, J. Tam, S. Reddy, N.J. Webster, Muscleblind-like 1 (Mbnl1) promotes insulin receptor exon 11 inclusion via binding to a downstream evolutionarily conserved intronic enhancer, J. Biol. Chem. 285 (2010) 25426–25437. Y. Kino, C. Washizu, Y. Oma, H. Onishi, Y. Nezu, N. Sasagawa, N. Nukina, S. Ishiura, MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1, Nucleic Acids Res. 37 (2009) 6477–6490. E.S. Goers, J. Purcell, R.B. Voelker, D.P. Gates, J.A. Berglund, MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing, Nucleic Acids Res. 38 (2010) 2467–2484. D. Cass, R. Hotchko, P. Barber, K. Jones, D.P. Gates, J.A. Berglund, The four Zn fingers of MBNL1 provide a flexible platform for recognition of its RNA binding elements, BMC Mol. Biol. 12 (2011) 20. Ł.J. Sznajder, M. Michalak, K. Taylor, P. Cywoniuk, M. Kabza, A. WojtkowiakSzlachcic, M. Matłoka, P. Konieczny, K. Sobczak, Mechanistic determinants of MBNL activity, Nucleic Acids Res. 44 (2016) 10326–10342. Y. Kino, D. Mori, Y. Oma, Y. Takeshita, N. Sasagawa, S. Ishiura, Muscleblind protein, MBNL1/EXP, binds specifically to CHHG repeats, Hum. Mol. Genet. 13 (2004) 495–507. P. Konieczny, E. Stepniak-Konieczna, K. Sobczak, MBNL proteins and their target RNAs, interaction and splicing regulation, Nucleic Acids Res. 42 (2014) 10873–10887. A. Mykowska, K. Sobczak, M. Wojciechowska, P. Kozlowski, W.J. Krzyzosiak, CAG repeats mimic CUG repeats in the misregulation of alternative splicing, Nucleic Acids Res. 39 (2011) 8938–8951. K. Sobczak, G. Michlewski, M. de Mezer, E. Kierzek, J. Krol, M. Olejniczak, R. Kierzek, W. Krzyzosiak, Structural diversity of triplet repeat RNAs, J. Biol. Chem. 285 (2010) 12755–12764. H. Du, M.S. Cline, R.J. Osborne, D.L. Tuttle, T.A. Clark, J.P. Donohue, M.P. Hall, L. Shiue, M.S. Swanson, C.A. Thornton, M. Ares Jr., Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy, Nat. Struct. Mol. Biol. 17 (2010) 187–193. E.T. Wang, N.A. Cody, S. Jog, M. Biancolella, T.T. Wang, D.J. Treacy, S. Luo, G.P. Schroth, D.E. Housman, S. Reddy, et al., Transcriptome-wide regulation of premRNA splicing and mRNA localization by muscleblind proteins, Cell. 150 (2012) 710–724. C. Gooding, C. Edge, M. Lorenz, M.B. Coelho, M. Winters, C.F. Kaminski, D. Cherny, I.C. Eperon, C.W. Smith, MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3, Nucleic Acids Res. 41 (2013) 4765–4782. D. Pervouchine, Y. Popov, A. Berry, B. Borsari, A. Frankish, R. Guigó, Integrative transcriptomic analysis suggests new autoregulatory splicing events coupled with nonsense-mediated mRNA decay, Nucleic Acids Res. 47 (2019) 5293–5306 (pii: gkz193).

BBA - Gene Regulatory Mechanisms xxx (xxxx) xxxx

C.-S. Hung and J.-C. Lin

SRPK1 with molecular chaperones, Genes Dev. 23 (2009) 482–495. [57] P. Konieczny, E. Stepniak-Konieczna, K. Sobczak, MBNL expression in autoregulatory feedback loops, RNA Biol. 15 (2018) 1–8. [58] T. Tabaglio, D.H. Low, W.K.L. Teo, P.A. Goy, P. Cywoniuk, H. Wollmann, J. Ho, D. Tan, J. Aw, A. Pavesi, K. Sobczak, D.K.B. Wee, E. Guccione, MBNL1 alternative splicing isoforms play opposing roles in cancer, Life Sci. Alliance 1 (2018) e201800157. [59] F. Terenzi, A.N. Ladd, Conserved developmental alternative splicing of muscleblindlike (MBNL) transcripts regulates MBNL localization and activity, RNA Biol. 7 (2010) 43–55. [60] H. Tran, N. Gourrier, C. Lemercier-Neuillet, C.M. Dhaenens, A. Vautrin, F.J. Fernandez-Gomez, L. Arandel, C. Carpentier, H. Obriot, S. Eddarkaoui, L. Delattre, E. Van Brussels, I. Holt, G.E. Morris, B. Sablonnière, L. Buée, N. CharletBerguerand, S. Schraen-Maschke, D. Furling, I. Behm-Ansmant, C. Branlant, M.L. Caillet-Boudin, N. Sergeant, Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of Muscleblind-like-1 isoforms, J. Biol. Chem. 286 (2011) 16435–16446.

[51] Y. Sun, Y. Bao, W. Han, F. Song, X. Shen, J. Zhao, J. Zuo, D. Saffen, W. Chen, Z. Wang, X. You, Y. Wang, Autoregulation of RBM10 and cross-regulation of RBM10/RBM5 via alternative splicing-coupled nonsense-mediated decay, Nucleic Acids Res. 45 (2017) 8524–8540. [52] P. Konieczny, E. Stepniak-Konieczna, K. Taylor, L.J. Sznajder, K. Sobczak, Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript, Nucleic Acids Res. 45 (2017) 1760–1775. [53] Y. Taylor, C. Washizu, M. Kurosawa, Y. Oma, N. Hattori, S. Ishiura, N. Nukina, Nuclear localization of MBNL1: splicing-mediated autoregulation and repression of repeat-derived aberrant proteins, Hum. Mol. Genet. 24 (2015) 740–756. [54] D.P. Gates, L.A. Coonrod, J.A. Berglund, Autoregulated splicing of muscleblind-like 1 (MBNL1) pre-mRNA, J. Biol. Chem. 286 (2011) 34224–34233. [55] R. Sinha, E. Allemand, Z. Zhang, R. Karni, M.P. Myers, A.R. Krainer, Arginine methylation controls the subcellular localization and functions of the oncoprotein splicing factor SF2/ASF, Mol. Cell. Biol. 30 (2010) 2762–2774. [56] X.Y. Zhong, J.H. Ding, J.A. Adams, G. Ghosh, X.D. Fu, Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of

12