Studying Tissue Specific Diversity of Splicing Regulator Muscleblind

The muscleblind (mbl) family of RNA-binding proteins regulates alternative splicing, determining mRNA transcript composition for various types of tissue, and has been implicated in myotonic dystrophy. The mbl gene is subject to alternative splicing in Drosophila, leading to multiple isoforms, and has several paralogs in humans. Mbl proteins vary significantly in length, although the significance of such diversity and the role of specific isoforms have not been fully explored. Using immunofluorescence microscopy and polyclonal serum, we analyzed Mbl protein expression across adult Drosophila tissues. Mbl was detected in various locations, including the brain, gonads, muscle, and gut epithelium. Skeletal muscles demonstrated the greatest diversity in Mbl expression, with other tissues showing more homogenous expression. Mbl was present at low levels in flight and jump muscles, while other thoracic muscles and abdominal muscles showed high Mbl levels. Intracellular localization of Mbl was typically nuclear, however in the nervous system the protein was strongly expressed in the cytoplasm. During early adult development in the pupa, in various tissues Mbl was initially detected in discrete nuclear bodies, before it assumed more general nuclear staining. Our study reveals natural locations that have drastically different levels of Mbl as well as its intracellular localization. We conducted tissue specific molecular analysis of mbl transcript alternatively spliced exon regions using end point and reverse transcriptase qPCR, to determine whether mbl isoforms had preferential usage in differentiated tissue. Our molecular assays showed that brain tissue had stronger expression of exon 23 transcript relative to other exons of interest. Finally, we used fluorescent in situ hybridization probes to characterize expression of alternatively spliced exon transcripts across differentiation adult drosophila tissue. We found robust mbl expression in the central nervous system and variable exon presence in visceral and skeletal muscle tissue. Our findings suggest alternatively spliced mbl exons lead to isoforms with variable function and expression