Alternatively spliced MBNL1 isoforms exhibit differential influence on enhancing brown adipogenesis.

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.

Berberine Promotes Beige Adipogenic Signatures of 3T3-L1 Cells by Regulating Post-transcriptional Events.

Induced brown adipocytes (also referred to as beige cells) execute thermogenesis, as do the classical adipocytes by consuming stored lipids, being related to metabolic homeostasis. Treatment of phytochemicals, including berberine (BBR), was reported to induce conversion from white adipocytes to beige cells. In this study, results of microRNA (miRNA)-seq analyses revealed a decrease in miR-92a, of which the transcription is driven by the c13orf25 promoter in BBR-treated 3T3-L1 cells. BBR treatment manipulated the expressions of SP1 and MYC, in turn, reducing the activity of the c13orf25 promoter. A decrease in miR-92a led to an increase in RNA-binding motif protein 4a (RBM4a) expression, which facilitated the beige adipogenesis. Overexpression of miR-92a or depletion of RBM4a reversely interfered with the impact of BBR treatment on the beige adipogenic signatures, gene expressions, and splicing events in 3T3-L1 cells. Our findings demonstrated that BBR treatment enhanced beige adipogenesis of 3T3-L1 cells through transcription-coupled post-transcriptional regulation.

RBM4a modulates the impact of PRDM16 on development of brown adipocytes through an alternative splicing mechanism.

Brown adipocytes (BAs) exhibit an energy-expending signature that is important in balancing metabolic homeostasis. In this study, results of transcriptome analyses revealed the reprogrammed splicing profile of the PR domain containing 16 (PRDM16) gene, a key transcription factor involved in brown adipogenesis, throughout development of wild-type brown adipose tissues (BATs). Moreover, discriminative splicing patterns of PRDM16 transcripts were noted in embryonic and postnatal RBM4a-/- BATs. Overexpression of RBM4a enhanced the relative levels of PRDM16-ex 16 transcripts by simultaneously interacting with exonic and intronic CU elements, which encoded the PRDM16S isoform containing a distinct C-terminus. The presence of the overexpressed PRDM16S isoform showed a stronger effect than the overexpressed PRDM16L isoform on enhancing transcriptional activity of the RBM4a and the PGC-1α promoter. Overexpression of the PRDM16S isoform exerted more-prominent effects on enhancing the BAT-related gene program and energy expenditure compared to those of PRDM16L-overexpressing cells. Our studies demonstrated that RBM4a-regulated alternative splicing constituted another regulatory mechanism for strengthening the influence of PRDM16 on the development of brown adipocytes.

RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis.

An increase in mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) reportedly attenuates insulin-mediated signaling which participates in the development of brown adipose tissues (BATs). Nevertheless, the effect of MAP4K4 on brown adipogenesis remains largely uncharacterized. In this study, results of a transcriptome analysis (also referred as RNA-sequencing) showed differential expressions of MAP4K4 or SRSF3 transcripts isolated from distinct stages of embryonic BATs. The discriminative splicing profiles of MAP4K4 or SRSF3 were noted as well in brown adipocytes (BAs) with RNA-binding motif protein 4-knockout (RBM4-/-) compared to the wild-type counterparts. Moreover, the relatively high expressions of authentic SRSF3 transcripts encoding the splicing factor functioned as a novel regulator toward MAP4K4 splicing during brown adipogenesis. The presence of alternatively spliced MAP4K4 variants exerted differential effects on the phosphorylation of c-Jun N-terminal protein kinase (JNK) which was correlated with the differentiation or metabolic signature of BAs. Collectively, the RBM4-SRSF3-MAP4K4 splicing cascade constitutes a novel molecular mechanism in manipulating the development of BAs through related signaling pathways.

RBM4-Nova1-SRSF6 splicing cascade modulates the development of brown adipocytes.

Alternative splicing (AS) is a pivotal mechanism for the expansion of gene diversity, which determines the cellular fate or specification. However, the effect of AS networks on brown adipogenesis has not been comprehensively investigated. In this study, we identified the discriminative splicing profiles of RNA-binding motif protein 4a-knockout (RBM4a-/-) brown adipocytes (BAs) and compared them with those of their wild-type counterparts through deep RNA-sequencing. Among these candidates, RBM4a ablation enhanced the relative level of exon 4-excluded neuro-oncological ventral antigen 1 (Nova1-4) transcripts, which were predominantly generated in embryonic BAs. By contrast, most of the Nova1 transcripts were exon 4-included (Nova1+4) in mature BAs. The Nova1 isoforms exhibited differential effects on repressing the development of BAs. Moreover, overexpression of Nova1 proteins reduced the serine/arginine splicing factor 6 (SRSF6) level by enhancing the generation of intron 2-included (SRSF6+intron 2) transcripts, which are a putative candidate of the AS-coupled nonsense-mediated decay mechanism. Furthermore, we observed the positive effect of SRSF6 on BA development. These results highlight the hierarchical role of RBM4a in an AS cascade that manipulates brown adipogenesis.