Alternative splicing is a major form of post-transcriptional regulation of gene expression which plays a pivotal role in cell/tissue- specific identity, diversity, and function. The mechanisms of alternative splicing are well studied; however, the functional consequences of the spliced transcripts are relatively less demonstrated. Therefore, many efforts are needed to understand the physiological relevance of alternative splicing. RNA binding proteins are critical regulators of alternative splicing, their malfunction disrupt the normal networks, and further underlies many diseases. To understand how alternative splicing contributes to the physiological functions in the cells or tissues, we explored the regulatory networks of RNA binding protein MEC-8/RBPMS, aim to identify the functionally relevant targets and characterize the RNA map of MEC-8/RBPMS in vivo.
MEC-8/RBPMS is a conserved splicing factor and is vital in various function in Caenorhabditis elegans, one example is its requirement for the mechanosensation in touch neurons. However, how MEC-8 contributes to the function of touch neurons is poorly understood. In this work, by using a combination of cell-specific transcriptomics, gene editing and behavioral analysis, we identified a small MEC-8-regulated network in touch neurons, where MEC-8 establishes two mec-2 touch neuron-specific isoforms (mec-2A and mec-2E) differing from default isoforms (mec-2B) found in other cells. Both mec-2 touch neuron-specific isoforms are simultaneously needed for touch neuron function, likely functioning as heteromers to mediate mechanosensation. Furthermore, we found mec-2B expresses in specific olfactory neurons and is required for olfaction. Together, we elucidated the MEC-8-regulated specific splicing events of mec-2 is vital for mechanosensation in touch neurons, provided a good example to demonstrate the importance of alternative splicing for proper function of the cells.
Next, we further explored the olfactory function of mec-2, which has not been reported before. By analyzing cell-specific transcriptomic data and using olfactory assays, we showed mec-2 is the only stomatin domain gene required for olfaction in C. elegans, though other stomatin domain genes are expressed in olfactory neurons. Further, we extended our finding, the olfaction role for mec-2, to the mouse homologue Stoml3. We generated Stoml3 knockout mice, performed a set of olfactory assays and demonstrated that, like its worm homologue mec-2, Stoml3 is required for olfactory behavior. Combined with previous studies, in addition to their shared roles in mechanosensory behavior, our work revealed mec-2 and stoml3 also have a shared role in olfactory behavior.
Last, we defined transcriptome-wide RNA targets of MEC-8 by using crosslinking immunoprecipitation sequencing (CLIP-Seq) in C. elegans. Combining alternative splicing events regulated by MEC-8 from RNA-Seq data, we revealed a set of direct regulatory targets of MEC-8 in whole animals. Moreover, using bioinformatic tools we denoted the binding feature and enriched binding motifs of MEC-8 at nucleotide resolution in vivo. Altogether, the RNA map of MEC-8 inferred potential roles of these targets for cell and tissue function. Our work significantly facilitates the understanding of how splicing factors like MEC-8 contributes to the identify and function of the cell and tissue.
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Liang, Xiaoyu, "Regulation and Function of the MEC-8/RBPMS RNA Binding Protein in vivo" (2022). Biological Sciences Theses and Dissertations. 18.