The Neural RNA-Binding Protein Musashi1 Translationally Regulates Mammalian numb Gene Expression by Interacting with Its mRNA

Abstract

Musashi1 (Msi1) is an RNA-binding protein that is highly expressed in neural progenitor cells, including neural stem cells. In this study, the RNA-binding sequences for Msi1 were determined by in vitro selection using a pool of degenerate 50-mer sequences. All of the selected RNA species contained repeats of (G/A)U _n AGU (n = 1 to 3) sequences which were essential for Msi1 binding. These consensus elements were identified in some neural mRNAs. One of these, mammaliannumb (m-numb), which encodes a membrane-associated antagonist of Notch signaling, is a likely target of Msi1. Msi1 protein binds in vitro-transcribed m-numb RNA in its 3′-untranslated region (UTR) and binds endogenousm-numb mRNA in vivo, as shown by affinity precipitation followed by reverse transcription-PCR. Furthermore, adenovirus-induced Msi1 expression resulted in the down-regulation of endogenous m-Numb protein expression. Reporter assays using a chimeric mRNA that combined luciferase and the 3′-UTR of m-numb demonstrated that Msi1 decreased the reporter activity without altering the reporter mRNA level. Thus, our results suggested that Msi1 could regulate the expression of its target gene at the translational level. Furthermore, we found that Notch signaling activity was increased by Msi1 expression in connection with the posttranscriptional down-regulation of the m-numb gene.

ACKNOWLEDGMENTS

We are grateful to Mikiko Siomi and Haruhiko Siomi for kind instructions for subcellular fractionation using sucrose density gradients and to Yuh Nung Jan for providing us with the m-numb cDNA. We thank Keiko Nakao-Sawai, Wado Akamatsu, Shinichi Sakakibara, and Hirotaka James Okano for valuable discussions and comments and Yoshio Wakamatsu for providing us with the anti-chicken-Numb antiserum and for the valuable suggestion regarding the 3′-UTR sequence of chick and human numb.

This work was supported by grants to H.O. from the Japanese Ministry of Education, Science and Culture and from CREST, Japan and Technology Corporation. T.I. was a research fellow of the Japan Society for the Promotion of Science. This work was also supported by grants from the Human Frontier Science Program to H.O.