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Molecular and physiological roles of long 3′ UTR mRNA isoforms in neurons
AuthorBae, Bong Min
Cell and Molecular Biology
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The brain is an organ where the greatest proportion of genes are expressed compared to any other part of the body. To add even more complexity, gene expression in the brain is subject to various layers of regulation through RNA processing mechanisms including alternative splicing (AS) and alternative cleavage and polyadenylation (APA). These RNA processing mechanisms contribute to increased transcriptome diversity in the brain. APA often induces the synthesis of mRNA isoforms that harbor the same protein-coding sequence but different length 3′ untranslated regions (3′ UTRs) from a single gene. Alternative 3′ UTRs regulate gene expression post-transcriptionally by modulating transcript stability, translation efficiency, or subcellular localization. In Chapter 1, we reviewed all of the reported functions of 3′ UTRs in the nervous system. Despite the fact 3′ UTR is highly regarded in gene regulation, evidence of impacts of long 3′ UTR loss on in vivo animal is scarce. To study the physiological relevance of long 3′ UTR mRNA isoforms, we have driven our attention to the Calm1 gene. Calm1 is one of the three genes that encode Calmodulin which is required for proper neural development and function. In Chapter 2, we found that the expression of the long 3′ UTR mRNA isoform of Calm1 was necessary for mouse nervous system development and function. Disruption of the Calm1 long 3′ UTR isoform impaired dorsal root ganglion axon development in mouse embryos and neuronal activation upon novel environment exposure in young adult mice. Our results presented direct evidence for the physiological importance of the Calm1 long 3′ UTR mRNA isoform in vivo. To screen molecular and cellular functions of long 3′ UTRs in a fast and efficient manner, establishing an in vitro cell system is warranted. In Chapter 3, we presented mouse embryonic stem cell (mESC)-derived neurons as a suitable cell-culture system. The transcriptomic profile of the mESC-derived neurons closely resembled the profile in the mouse cerebral cortex, showing the suitability of using this system for studying long 3′ UTRs. The mESC system is amenable to genetic manipulation via CRISPR-Cas9, thus providing as good avenue for fast generation of long 3′ UTR isoform knockout lines. As a proof of principle, a workflow for the generation of Myosin phosphatase Rho interacting protein (Mprip) long 3′ UTR isoform knockout cell lines, differentiation into glutamatergic neurons, and confirmation of the long 3′ UTR expression abolishment is presented. Taking advantage of the convenient culture cell system we have established, we next aimed to explore more functions of long 3′ UTRs. A recent discovery in our lab suggested that APA and AS are closely linked RNA processing mechanisms in which long 3′ UTRs modulate upstream AS. In Chapter 4, we explored the coupling events between AS and APA in mouse neurons using Pull-a-Long-Seq (PL-Seq) pipeline, which presents a particular utility in quantifying the coordination of tandem 3′ UTR APA events with upstream cassette exon AS. PL-Seq performed on the Endonuclease V (Endov) gene reveals that expression of its long 3′ UTR in neurons is preferentially associated with an exon skipping event located far upstream of the terminal exon.