An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.     

Asymmetric sorting of ash1p in yeast results from inhibition of translation by localization elements in the mRNA

ASH1 mRNA localizes at the bud tip of late-anaphase yeast, resulting in accumulation of Ash1p in the daughter nucleus. We show that disruption of the secondary structure, but not the protein coding, of all four ASH1 localization elements resulted in RNA and protein delocalization. Localization of both was incrementally restored by replacement of each of the four elements. However, transposition of the elements to the 3'UTR reinstated the RNA, but not the protein, localization. Interestingly, the mutant ASH1 mRNA was translated more efficiently, suggesting that asymmetry of Ash1p resulted from translational inhibition by the localization elements. In support of this, Ash1p asymmetry could be rescued by slowing its translation.     

Stability control of MTL1 mRNA by the RNA-binding protein Khd1p in yeast

Khd1p (KH-domain protein 1) is a yeast RNA-binding protein highly homologous to mammalian hnRNP K. Khd1p associates with hundreds of potential mRNA targets including a bud-localized ASH1 mRNA and mRNAs encoding membrane-associated proteins such as Mid2p and Mtl1p. While Khd1p negatively regulates gene expression of Ash1p by translational repression, Khd1p positively regulates gene expression of Mtl1p by mRNA stabilization. To investigate how Khd1p regulates the stability of MTL1 mRNA, we searched for cis-acting elements and trans-acting factors controlling MTL1 mRNA stability. Regional analysis revealed that partial deletion of the coding sequences of MTL1 mRNA restored the decreased MTL1 mRNA and protein levels in khd1Δ mutants. This region, encompassing nucleotides 532 to 1032 of the Mtl1p coding sequence, contains CNN repeats that direct Khd1p-binding. Insertion of this sequence into other mRNAs conferred mRNA instability in khd1Δ mutants. We further searched for factors involved in the destabilization of MTL1 mRNA. Mutations in CCR4 and CAF1/POP2, encoding major cytoplasmic deadenylases, or of SKI genes, which code for components of a complex involved in 3' to 5' degradation, did not restore the decreased MTL1 mRNA levels caused by khd1Δ mutation. However, mutations in DCP1 and DCP2, encoding a decapping enzyme complex, and XRN1, encoding a 5'-3' exonuclease, restored the decreased MTL1 mRNA levels. Furthermore, Khd1p colocalized with Dcp1p in processing bodies, cytoplasmic sites for mRNA degradation. Our results suggest that MTL1 mRNA bears a cis-acting element involved in destabilization by the decapping enzyme and the 5'-3' exonuclease, and Khd1p stabilizes MTL1 mRNA through binding to this element.     

ASH1 mRNA localization in yeast involves multiple secondary structural elements and Ash1 protein translation

Localization of ASH1 mRNA to the distal cortex of daughter but not mother cells at the end of anaphase is responsible for the two cells' differential mating-type switching during the subsequent cell cycle. This localization depends on actin filaments and a type V myosin (She1/Myo4). The 3' untranslated region (3' UTR) of ASH1 mRNA is reportedly capable of directing heterologous RNAs to a mother cell's bud [1] [2]. Surprisingly, however, its replacement has little or no effect on the localisation of ASH1 mRNA. We show here that, unlike all other known localization sequences that have been found in 3' UTRs, all the elements involved in ASH1 mRNA localization are located at least partly within its coding region. A 77 nucleotide region stretching from 7 nucleotides 5' to 67 nucleotides 3' of the stop codon of ASH1 mRNA is sufficient to localize mRNAs to buds; the secondary structure of this region, in particular two stems, is important for its localizing activity. Two regions entirely within coding sequences, both sufficient to localize green fluorescent protein (GFP) mRNA to growing buds, are necessary for ASH1 mRNA localization during anaphase. These three regions can anchor GFP mRNA to the distal cortex of daughter cells only inefficiently. The tight anchoring of ASH1 mRNA to the cortex of the daughter cell depends on translation of the carboxy-terminal sequences of Ash1 protein.     

RNA localization in yeast: moving towards a mechanism

RNA localization is a widely utilized strategy employed by cells to spatially restrict protein function. In Saccharomyces cerevisiae asymmetric sorting of mRNA to the bud has been reported for at least 24 mRNAs. The mechanism by which the mRNAs are trafficked to the bud, illustrated by ASH1 mRNA, involves recognition of cis-acting localization elements present in the mRNA by the RNA-binding protein, She2p. The She2p/mRNA complex subsequently associates with the myosin motor protein, Myo4p, through an adapter, She3p. This ribonucleoprotein complex is transported to the distal tip of the bud along polarized actin cables. While the mechanism by which ASH1 mRNA is anchored at the bud tip is unknown, current data point to a role for translation in this process, and the rate of translation of Ash1p during the transport phase is regulated by the cis-acting localization elements. Subcellular sorting of mRNA in yeast is not limited to the bud; certain mRNAs corresponding to nuclear-encoded mitochondrial proteins are specifically sorted to the proximity of mitochondria. Analogous to ASH1 mRNA localization, mitochondrial sorting requires cis-acting elements present in the mRNA, though trans-acting factors involved with this process remain to be identified. This review aims to discuss mechanistic details of mRNA localization in S. cerevisiae.     

RNA-protein interactions promote asymmetric sorting of the ASH1 mRNA ribonucleoprotein complex

In Saccharomyces cerevisiae, ASH1 mRNA is localized to the tip of daughter cells during anaphase of the cell cycle. ASH1 mRNA localization is dependent on four cis-acting localization elements as well as Myo4p, She2p, and She3p. Myo4p, She2p, and She3p are hypothesized to form a heterotrimeric protein complex that directly transports ASH1 mRNA to daughter cells. She2p is an RNA-binding protein that directly interacts with ASH1 cis-acting localization elements and associates with She3p. Here we report the identification of seven She2p mutants-N36S, R43A, R44A, R52A, R52K, R63A, and R63K-that result in the delocalization of ASH1 mRNA. These mutants are defective for RNA-binding activity but retain the ability to interact with She3p, indicating that a functional She2p RNA-binding domain is not a prerequisite for association with She3p. Furthermore, the nuclear/cytoplasmic distribution for the N36S and R63K She2p mutants is not altered, indicating that nuclear/cytoplasmic trafficking of She2p is independent of RNA-binding activity. Using the N36S and R63K She2p mutants, we observed that in the absence of She2p RNA-binding activity, neither Myo4p nor She3p is asymmetrically sorted to daughter cells. However, in the absence of She2p, Myo4p and She3p can be asymmetrically segregated to daughter cells by artificially tethering mRNA to She3p, implying that the transport and/or anchoring of the Myo4p/She3p complex is dependent on the presence of associated mRNA.     

Structural elements required for the localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo

The sorting of the Ash1 protein to the daughter nucleus of Saccharomyces cerevisiae in late anaphase of the budding cycle correlates with the localization of ASH1 mRNA at the bud tip [1] [2]. Although the 3' untranslated region (3' UTR) of ASH1 is sufficient to localize a reporter mRNA, it is not necessary, a result which indicates that other sequences are involved [1]. We report the identification of three additional cis-acting elements in the coding region. Each element alone, when fused to a lacZ reporter gene, was sufficient for the localization of the lacZ mRNA reporter to the bud. A fine-structure analysis of the 3' UTR element showed that its function in mRNA localization did not depend on a specific sequence but on the secondary and tertiary structure of a minimal 118 nucleotide stem-loop. Mutations in the stem-loop that affect the localization of the lacZ mRNA reporter also affected the formation of the localization particles, in living cells, composed of a green fluorescent protein (GFP) complexed with lacZ-ASH1-3' UTR mRNA [3]. A specific stem-loop in the 3' UTR of the ASH1 mRNA is therefore required for both localization and particle formation, suggesting that complex formation is part of the localization mechanism. An analysis on one of the coding-region elements revealed a comparable stem-loop structure with similar functional requirements.     

RNA asymmetric distribution and daughter/mother differentiation in yeast

Active transport and localized translation of the ASH1 mRNA at the bud tip of the budding yeast Saccharomyces cerevisiae is an essential process that is required for the regulation of the mating type switching. ASH1 mRNA localization has been extensively studied over the past few years and the core components of the translocation machinery have been identified. It is composed of four localization elements (zipcodes), within the ASH1 mRNA, and at least three proteins, She1p/Myo4p, She2p and She3p. Whereas the movement of the RNA can be attributed to direct interaction with myosin, the regulation of the RNA expression is less well understood. Recent insights have revealed a role for translation that might have a key function in the regulation of Ash1 protein sorting.