Abstract
The Musashi family of RNA binding proteins act to promote stem cell self-renewal and oppose cell differentiation predominantly through translational repression of mRNAs encoding pro-differentiation factors and inhibitors of cell cycle progression. During tissue development and repair however, Musashi repressor function must be dynamically regulated to allow cell cycle exit and differentiation. The mechanism by which Musashi repressor function is attenuated has not been fully established. Our prior work indicated that the Musashi1 isoform undergoes site-specific regulatory phosphorylation. Here, we demonstrate that the canonical Musashi2 isoform is subject to similar regulated site-specific phosphorylation, converting Musashi2 from a repressor to an activator of target mRNA translation. We have also characterized a novel alternatively spliced, truncated isoform of human Musashi2 (variant 2) that lacks the sites of regulatory phosphorylation and fails to promote translation of target mRNAs. Consistent with a role in opposing cell cycle exit and differentiation, upregulation of Musashi2 variant 2 was observed in a number of cancers and overexpression of the Musashi2 variant 2 isoform promoted cell transformation. These findings indicate that alternately spliced isoforms of the Musashi protein family possess distinct functional and regulatory properties and suggest that differential expression of Musashi isoforms may influence cell fate decisions.
Highlights
Targeted control of mRNA translation is gaining recognition as a key mechanism for regulation of cell cycle and cell fate transitions[1,2,3,4,5]
We demonstrate that Msi[2] phosphorylation is mediated by both Ringo/CDK signaling and p42 mitogen activated protein (MAP) kinase (ERK) signaling pathways and that mutational disruption of Msi[2] phosphorylation abrogates stimulus-dependent target mRNA translational activation and Xenopus oocyte maturation
Using RNA Electrophoretic mobility shift assay (EMSA), we confirmed that the hMsi[2] variant 2 was able to bind to a target mRNA with similar efficacy as the canonical hMsi[2] protein (Fig. 6d and e). These findings indicate that the inability of the alternatively spliced Msi[2] variant 2 isoform to mediate oocyte maturation is most likely due to the fact the Msi[2] variant 2 protein evades the C-terminal regulatory phosphorylation necessary for de-repression and/or activation of target mRNA translation
Summary
Targeted control of mRNA translation is gaining recognition as a key mechanism for regulation of cell cycle and cell fate transitions[1,2,3,4,5]. Consistent with possible shared mRNA targets, Msi[1] and Msi[2] have been shown to effectively substitute for each other in the control of Xenopus oocyte maturation, mammalian neuronal stem cell self-renewal, intestinal stem cell quiescence and colorectal cancer[32, 40,41,42]. Despite these apparent similarities, several lines of evidence suggest differences between the Msi family members, in terms of expression patterns, as well as interaction with protein binding partners and function. Our results indicate that the canonical Msi[1] and Msi[2] proteins have shared regulatory mechanisms while highlighting an unexpected role for alternative splicing in Msi[2] isoform-specific control
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