Gfi1 is a transcriptional repressor essential for haematopoiesis and inner ear development. It shares with its paralogue Gfi1b an amino-terminal SNAG repressor domain and six carboxy-terminal zinc-finger motifs, but differs from Gfi1b in sequences separating these domains. Here, we describe two knock-in mouse models, in which the N-terminal SNAG repressor domain was mutated or in which the Gfi1 coding region was replaced by Gfi1b. Mouse mutants without an intact SNAG domain show the full phenotype of Gfi1 null mice. However, Gfi1:Gfi1b knock-in mice show almost normal pre-T-cell and neutrophil development, but lack properly formed inner ear hair cells. Hence, our findings show that an intact SNAG domain is essential for all functions of Gfi1 and that Gfi1b can replace Gfi1 functionally in haematopoiesis but, surprisingly, not in inner ear hair cell development, demonstrating that Gfi1 and Gfi1b have equivalent and domain-dependent, cell type-specific functions.
The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). Identification of the interacting proteome of a specific mRNA in vivo remains very challenging, however. Based on the widely used technique of RNA tagging with MS2 aptamers for RNA visualization, we developed a RNA proximity biotinylation (RNA-BioID) technique by tethering biotin ligase (BirA*) via MS2 coat protein at the 3′ UTR of endogenous MS2-tagged β-actin mRNA in mouse embryonic fibroblasts. We demonstrate the dynamics of the β-actin mRNA interactome by characterizing its changes on serum-induced localization of the mRNA. Apart from the previously known interactors, we identified more than 60 additional β-actin–associated RBPs by RNA-BioID. Among these, the KH domain-containing protein FUBP3/MARTA2 has been shown to be required for β-actin mRNA localization. We found that FUBP3 binds to the 3′ UTR of β-actin mRNA and is essential for β-actin mRNA localization, but does not interact with the characterized β-actin zipcode element. RNA-BioID provides a tool for identifying new mRNA interactors and studying the dynamic view of the interacting proteome of endogenous mRNAs in space and time.
Protein translocation into the endoplasmic reticulum (ER) generally requires targeting of mRNAs encoding secreted or membrane proteins to the ER membrane. The prevalent view is that these mRNAs are delivered co-translationally, using the signal recognition particle (SRP) pathway. Here, SRP delivers signal sequence-containing proteins together with associated ribosomes and mRNA to the SRP receptor present on the ER surface. Recent studies demonstrate the presence of alternative pathways to recruit mRNAs to ER or to specific subdomains of the ER independent of SRP or translation. Such targeting of specific mRNAs to the ER subdomains allows the cell to sort proteins before translocation or to ensure co-localization of ER and mRNAs at specific locations. Translation-independent association of mRNAs involves ER-linked RNA-binding proteins and represents an alternative pathway of mRNA delivery to the ER. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.
Localization of messenger RNA (mRNAs) contributes to generation and maintenance of cellular asymmetry, embryonic development and neuronal function. The She1-3 protein machinery in Saccharomyces cerevisiae localizes >30 mRNAs to the bud tip, including 13 mRNAs encoding membrane or secreted proteins. Ribonucleoprotein (RNP) particles can co-localize with tubular endoplasmic reticulum (ER) structures that form the initial elements for segregation of cortical ER (cER), suggesting a coordination of mRNA localization and cER distribution. By investigating localization of MS2-tagged mRNAs in yeast defective at various stages of cER segregation, we demonstrate that proper cER segregation is required for localization of only a subset of mRNAs. These mRNAs include WSC2, IST2, EAR1 and SRL1 that encode membrane or ER associated proteins and are expressed during S and G2 phases of the cell cycle when tubular ER movement into the bud occurs. Translation of WSC2 is not required for localization, ruling out co-translational targeting of this mRNA. Localization of ASH1 mRNA is independent of cER segregation, which is consistent with the expression pattern of ASH1 at late mitosis. Our findings indicate the presence of two different pathways to localize mRNAs to the yeast bud.
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