MmuEX0028097 @ mm9

In this study, the authors 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 (MBNL1-7) to MBNL1-ex 5 isoform 1 (MBNL1-1) 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 MBNL1-1 isoform exhibited a more-prominent effect than that of the MBNL1-7 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 MBNL1-1 and MBNL1-7 isoforms differentially enhanced beige adipogenic signatures of 3T3-L1 cells.

The exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1

The presence of alternative ex.54nt always significantly elevated MBNL1 and MBNL2 protein levels (_2.7 and _4.0-times, respectively). There were marginal changes of the GFP-MBNL level for constructs carrying ex.36nt (_1.3-times) and ex.95nt (_0.8-times). There were no significant differences between the activities of the analyzed proteins for the majority (67%) of AS events. However, these exons might have either a positive or negative effect on some specific AS events (Figure 5A and C). Interestingly, exOFF events predominated in AS events with a negative effect of ex.36nt (95%; P = 0.002) and a positive effect of ex.95nt (90%; P = 0.010). All together, these data indicate that the presence of sequences encoded by these alternative exons can significantly modulate MBNL splicing activity, but this regulation depends strongly on the targeted RNA.

Inclusion/exclusion of the exon affects splicing activity with sequential and overlapping binding motifs, as shown by competition assays between isoforms.

Nuclear localization. Induction of MBNL1 exon skipping drove expected functional consequences. Nuclear levels of total MBNL1 were quantitatively lower following delivery of each pgRNA that induced appreciable exon skipping.