In hippocampal neurons, certain mRNAs have been found in dendrites (Steward, 1997), and their localization and translation have been implicated in synaptic plasticity (Martin et al., 1997). One attractive candidate to achieve transport of mRNAs into dendrites is Staufen (Stau), a double-stranded RNA-binding protein, which plays a pivotal role in mRNA transport, localization, and translation in Drosophila (St. Johnston, 1995). Using antibodies raised against a peptide located in the RNA-binding domain IIa and a polyclonal antibody raised against a recently cloned human Staufen homolog, we identify a 65 kDa rat homolog in cultured rat hippocampal neurons. In agreement with the exclusive somatodendritic localization of mRNAs in these cells, we find that Staufen is restricted to the same domain. By immunoelectron microscopy, we show enrichment of the mammalian homolog of Stau (mStau) in the vicinity of smooth endoplasmic reticulum and microtubules near synaptic contacts. Finally, the association of the mStau with neuronal mRNAs is suggested by the colocalization with ribonucleoprotein particles specifically in distal dendrites known to contain mRNA, ribosomes, and translation factors (Knowles et al., 1996). These results suggest a role for mStau in the polarized transport and localization of mRNAs in mammalian neurons.

In the course of a two-hybrid screen with the NS1 protein of influenza virus, a human clone capable of coding for a protein with high homology to the Staufen protein from Drosophila melanogaster (dmStaufen) was identified. With these sequences used as a probe, cDNAs were isolated from a cDNA library. The encoded protein (hStaufen-like) contained four double-stranded RNA (dsRNA)-binding domains with 55% similarity and 38% identity to those of dmStaufen, including identity at all residues involved in RNA binding. A recombinant protein containing all dsRNA-binding domains was expressed in Escherichia coli as a His-tagged polypeptide. It showed dsRNA binding activity in vitro, with an apparent K d of 10 ؊9 M. Using a specific antibody, we detected in human cells a major form of the hStaufen-like protein with an apparent molecular mass of 60 to 65 kDa. The intracellular localization of hStaufen-like protein was investigated by immunofluorescence using a series of markers for the cell compartments. Colocalization was observed with the rough endoplasmic reticulum but not with endosomes, cytoskeleton, or Golgi apparatus. Furthermore, sedimentation analyses indicated that hStaufen-like protein associates with polysomes. These results are discussed in relation to the possible functions of the protein.The establishment and maintenance of asymmetries in certain cells implies the localized expression of many proteins, a property often enhanced by the localization of the corresponding mRNAs (for reviews, see references 2 and 47). The relevance of mRNA localization at precise sites of the cell in the definition of polarity of developing embryos has been documented in both Drosophila melanogaster and Xenopus laevis. Thus, the positions of gurken and oskar mRNAs in the fly oocyte define its dorsoventral axis (36) and the location of the pole plasm at the posterior pole (15), respectively. Likewise, the localization of the bicoid and nanos mRNAs at the anterior and posterior poles of the embryo, respectively, leads to the generation of two opposing gradients of their protein products and ultimately to the definition of the head, thorax, and abdomen of the embryo (50). In the case of X. laevis, several mRNAs, such as Xcat2, Xcat3, and Xlsirt, are directed to the germ plasm (34), while others, such as Vg1 and Xwnt11, accumulate at the vegetal cortex (30). By analogy to the D. melanogaster genes, these mRNAs are thought to play a role in defining patterns in the X. laevis embryo. In fact, a region of the Xcat2 protein shows sequence homology to the zinc finger domain of nanos protein (34).The specific localization of certain mRNAs to precise sites within the cell is not an exclusive property of germ cells or developing embryos. A number of observations indicate that some of the mRNAs of somatic cells are also localized at different sites within the cell. Thus, myelin-binding protein is translated in oligodendrocytes from free ribosomes on localized mRNA (51), and the microtubule-associated protein MAP2 is translated preferentially i...

Yeast strains lacking the yeast nuclear cap-binding complex (yCBC) are viable, although impaired in growth. We have taken advantage of this observation to carry out a genetic screen for components that show synthetic lethality (SL) with a cbp20-Delta cbp80-Delta double mutation. One set of SL interactions was due to mutations that were complemented by components of U1 small nuclear RNP (snRNP) and the yeast splicing commitment complex. These interactions confirm the role of yCBC in commitment complex formation. Physical interaction of yCBC with the commitment complex components Mud10p and Mud2p, which may directly mediate yCBC function, was demonstrated. Unexpectedly, we identified multiple SL mutations that were complemented by Cbf5p and Nop58p. These are components of the two major classes of yeast small nucleolar RNPs, which function in the maturation of rRNA precursors. Mutants lacking yCBC were found to be defective in rRNA processing. Analysis of the yCBC deletion phenotype suggests that this is likely to be due to a defect in the splicing of a subset of ribosomal protein mRNA precursors.

The mRNA cap structure is bound by either the nuclear (CBC) or the cytoplasmic (eIF4F) cap binding complex. Following mRNA export, CBC must be exchanged for eIF4F in the cytoplasm. It is not known how this exchange occurs or how this RNP remodeling event is integrated with mRNA function. Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction. Furthermore, we find that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding. These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.