In general terms, the project is advancing correctly for most Aims, and some important articles have been published. The project consists of four Aims, which can be summarized by four questions on the functional and regulatory architecture of the nested microexon programs:
(1) How are the different levels of the master regulators controlled in each cell type? Through the analysis of RNA-seq, ATAC-seq and H3K27ac ChIP-seq data we identified (i) a glucose-responsive, pancreas-specific superenhancer regulating SRRM3, and (ii) a weak enhancer in the first intron of SRRM3, also relevant for its expression (Juan-Mateu et al, Nat Metab 2023). We found that both regulatory elements contain human variants associated with pathophysiological conditions. At the experimental level, sorting fluorescent cells from our zebrafish reporter lines for pancreas is proven difficult, but we hope to obtain them for ATAC-seq from collaborators.
(2) How are the distinct sensitivities of microexons to Srrm3/4 genomically encoded? We have designed five minigene libraries, covering length, cis-regulatory rules and evolution, expanding what we have originally planned. These libraries were tested with different levels of Srrm3/4 expression. The results led to an unexpected conclusion: the sensitivities are mainly due to differences in the core splicing architecture of microexons and not to differences in binding and effect of SRRM4. The manuscript with these results has been posted in bioRxiv (Bonnal et al 2024) and it is in advance stages of peer review.
(3) What are the functional implications of the 'nestedness' of the microexon programs? This is the largest Aim and can be divided into pancreas, neural and retina sub-aims. For pancreas, we have already published a first study with the functional characterization of pancreas microexons (IsletMICs) using cell cultures and a constitutive mouse KO for Srrm3 (Juan-Mateu et al, Nat Metab 2023). We have also investigated the role of Srrm3 and Srrm4 in glucose homeostasis in zebrafish, with non-conclusive results for now. For the neural part, we have succeeded in completing and publishing our work on the functional characterization of the impact of a neural-specific microexon in DAAM1. It has been published in Nature Communications (Polinski et al 2025). In addition, we are preparing a small follow up to be submitted. The work on another neural-specific microexon candidate (Unc13b) is ongoing.. For the retina, we have already published a first manuscript corresponding to the first tasks (Ciampi et al, PNAS 2022) and have generated five retina-specific microexon (RetMIC) KOs in zebrafish, and have assayed them for vision defects.
(4) How does misregulation of the nested programs contribute to disease? We have performed multiple computational analyses with available and de novo data for the three nested programs. The key results are reported in the manuscripts mentioned above. We are now assessing the potential of antisense oligonucleotides to manipulate IsletMIC inclusion with the hope of using them in diabetogenic contexts.
