Polarized growth in the budding yeast Saccharomyces cerevisiae depends upon the asymmetric localization and enrichment of polarity and secretion factors at the membrane prior to budding. We examined how these factors (i.e., Cdc42, Sec4, and Sro7) reach the bud site and found that their respective mRNAs localize to the tip of the incipient bud prior to nuclear division. Asymmetric mRNA localization depends upon factors that facilitate ASH1 mRNA localization (e.g., the 3 untranslated region, She proteins 1 to 5, Puf6, actin cytoskeleton, and a physical association with She2). mRNA placement precedes protein enrichment and subsequent bud emergence, implying that mRNA localization contributes to polarization. Correspondingly, mRNAs encoding proteins which are not asymmetrically distributed (i.e., Snc1, Mso1, Tub1, Pex3, and Oxa1) are not polarized. Finally, mutations which affect cortical endoplasmic reticulum (ER) entry and anchoring in the bud (myo4⌬, sec3⌬, and srp101) also affect asymmetric mRNA localization. Bud-localized mRNAs, including ASH1, were found to cofractionate with ER microsomes in a She2-and Sec3-dependent manner; thus, asymmetric mRNA transport and cortical ER inheritance are connected processes in yeast.

Subcellular mRNA localization, a fundamental mechanism for regulating gene expression, leads to local protein translation that results in the generation of neuronal cell polarity. In this study, we have used P19 embryonic carcinoma cells, which are amenable to transfection, and selection of clonal stable cell lines that are not overexpressing the constructs. We identified the 3Ј untranslated region (3ЈUTR) tau axonal localization signal and examined its effect on tau protein localization in nondifferentiated and neuronally differentiated P19 cells. Using GFPtagged tau constructs combined with in situ hybridization analysis, we demonstrated colocalization of the targeted tau mRNA and its translated protein in the axon and growth cone. Absence of or mutation in the 3ЈUTR axonal targeting region of tau mRNA resulted in suppression of tau mRNA localization, and both tau mRNA and tau protein remained in the cell body. Swapping between the 3ЈUTR tau mRNA axonal localization signal and the 3ЈUTR MAP2 mRNA dendritic targeting signal proved that the localization of the proteins into the axon or dendrites depends on the specific 3ЈUTR targeting signals. Moreover, the identification of ribosomal proteins in the axon lends further support to the presence of protein synthetic machinery in the axons, a prerequisite for local translation. It is suggested therefore that the P19 cell system can be used to analyze mutations that affect mRNA transport and local translation and that it has the potential of being used to examine the onset of the neuronal differentiation process. Key words: tau protein; tau mRNA; axonal targeting signal; ribosomes; P19 EC cells; neuronal differentiationNeuronal polarity results from the segregated distribution of molecules and organelles and depends on cytoskeletal organization (Bassell and Singer, 1997;Kiebler et al., 1999). It has been established that MAP2, the high molecular-weight microtubuleassociated protein (MAP), is localized in the cell body and dendrites, whereas tau MAP is found mainly in the cell body and axons (Matus et al., 1981;Binder et al., 1985).The molecular mechanisms responsible for the segregation of MAP proteins into the axons and dendrites are not yet fully understood. Subcellular mRNA localization and local translation within dendrites and axons are posttranscriptional control mechanisms that can explain this segregation and may play a key role in generation and maintenance of neuronal polarity. Recent data, which were obtained using molecular approaches and visualization, have demonstrated the presence of unique mRNA species and local protein synthesis in the dendrites, axons, and their growth cones

mRNA localization may be an important determinant for protein localization. We describe a simple PCR-based genomic-tagging strategy (m-TAG) that uses homologous recombination to insert binding sites for the RNA-binding MS2 coat protein (MS2-CP) between the coding region and 3' untranslated region (UTR) of any yeast gene. Upon coexpression of MS2-CP fused with GFP, we demonstrate the localization of endogenous mRNAs (ASH1, SRO7, PEX3 and OXA1) in living yeast (Saccharomyces cerevisiae).

Localization of tau mRNA to the axon requires the axonal localization cis signal (ALS), which is located within the 3′ untranslated region, and trans-acting binding proteins, which are part of the observed granular structures in neuronal cells. In this study, using both biochemical and morphological methods, we show that the granules contain tau mRNA, HuD RNA-binding protein, which stabilizes mRNA, and KIF3A, a member of the kinesin microtubuleassociated motor protein family involved in anterograde transport. The granules are detected along the axon and accumulate in the growth cone. Inhibition of KIF3A expression caused neurite retraction and inhibited tau mRNA axonal targeting. Taken together, these results suggest that HuD and KIF3A proteins are present in the tau mRNA axonal granules and suggest an additional function for the kinesin motor family in the microtubuledependent translocation of RNA granules. Localized tau-GFP expression was blocked by a protein synthesis inhibitor, and upon release from inhibition, nascent tau-GFP 'hot spots' were directly observed in the axon and growth cones. These observations are consistent with local protein synthesis in the axon resulting from the transported tau mRNA.

Both the delivery of secretory vesicles and asymmetric distribution of mRNA to the bud are dependent upon the actin cytoskeleton in yeast. Here we examined whether components of the exocytic apparatus play a role in mRNA transport. By screening secretion mutants in situ and in vivo, we found that all had an altered pattern of ASH1 mRNA localization. These included alleles of CDC42 and RHO3 (cdc42-6 and rho3-V51) thought to regulate specifically the fusion of secretory vesicles but were found to affect strongly the cytoskeleton as well. Most interestingly, mutations in late secretion-related genes not directly involved in actin regulation also showed substantial alterations in ASH1 mRNA distribution. These included mutations in genes encoding components of the exocyst (SEC10 and SEC15), SNARE regulatory proteins (SEC1, SEC4, and SRO7), SNAREs (SEC9 and SSO1/2), and proteins involved in Golgi export (PIK1 and YPT31/32). Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport. Our novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization.The establishment of cell polarity in eukaryotes involves the asymmetric organization of mRNA, the cytoskeleton, and the secretory pathway to lead to the polarized distribution of new membrane along a given axis (1, 2). In yeast, polarization leads to the budding of daughter cells (cell division), the asymmetric segregation of cell-fate determinants, and mating of haplotypes (3-4). These aspects of polarization require proper control of the actin cytoskeleton, as mutations therein block the exocytosis of proteins and new membrane along the axis of growth as well as the delivery of an mRNA encoding a protein involved in mating-type control (e.g. Ash1). For example, loss-of-function mutations in genes encoding yeast actin (ACT1), tropomyosin (TPM1,2), and a type V myosin (MYO2) all block exocytosis and result in lethality (5-7). Similarly, mutations in actin, tropomyosin, and another type V myosin (encoded by MYO4) also block ASH1 mRNA transport (2, 8 -11). Thus, an essentially common mechanism for both vesicle and mRNA transport to the growing bud appears to have evolved in eukaryotes.