Spatial regulation of translation through RNA localization.

RNA localization is a mechanism to post-transcriptionally regulate gene expression. Eukaryotic organisms ranging from fungi to mammals localize mRNAs to spatially restrict synthesis of specific proteins to distinct regions of the cytoplasm. In this review, we provide a general summary of RNA localization pathways in Saccharomyces cerevisiae, Xenopus, Drosophila and mammalian neurons.

Techniques for following the movement of single RNAs in living cells.

The ability to investigate gene expression has evolved from static approaches that analyze a population of cells to dynamic approaches that analyze individual living cells. During the last decade, a number of different fluorescent methods have been developed for monitoring the dynamics of single RNAs in living cells. Spatial-temporal analyses of single RNAs in living cells have provided novel insight into nuclear transport, RNA localization, and decay. Technical advances with these approaches allow for single molecule detection, providing an unprecedented view of RNA movement. In this article, we discuss the methods for observing single RNAs in living cells, highlighting the advantages and limitations of each method.

She3p possesses a novel activity required for ASH1 mRNA localization in Saccharomyces cerevisiae.

Intracellular and intercellular polarity requires that specific proteins be sorted to discreet locations within and between cells. One mechanism for sorting proteins is through RNA localization. In Saccharomyces cerevisiae, ASH1 mRNA localizes to the distal tip of the bud, resulting in the asymmetric sorting of the transcriptional repressor Ash1p. ASH1 mRNA localization requires four cis-acting localization elements and the trans-acting factors Myo4p, She3p, and She2p. Myo4p is a type V myosin motor that functions to directly transport ASH1 mRNA to the bud. She2p is an RNA-binding protein that directly interacts with the ASH1 mRNA cis-acting elements. Currently, the role for She3p in ASH1 mRNA localization is as an adaptor protein, since it can simultaneously associate with Myo4p and She2p. Here, we present data for two novel mutants of She3p, S348E and the double mutant S343E S361E, that are defective for ASH1 mRNA localization, and yet both of these mutants retain the ability to associate with Myo4p and She2p. These observations suggest that She3p possesses a novel activity required for ASH1 mRNA localization, and our data imply that this function is related to the ability of She3p to associate with ASH1 mRNA. Interestingly, we determined that She3p is phosphorylated, and global mass spectrometry approaches have determined that Ser 343, 348, and 361 are sites of phosphorylation, suggesting that the novel function for She3p could be negatively regulated by phosphorylation. The present study reveals that the current accepted model for ASH1 mRNA localization does not fully account for the function of She3p in ASH1 mRNA localization.

Nonsense-mediated decay of ash1 nonsense transcripts in Saccharomyces cerevisiae.

Nonsense-mediated mRNA decay (NMD) performs two functions in eukaryotes, one in controlling the expression level of a substantial subset of genes and the other in RNA surveillance. In the vast majority of genes, nonsense mutations render the corresponding transcripts prone to surveillance and subject to rapid degradation by NMD. To examine whether some classes of nonsense transcripts escape surveillance, we asked whether NMD acts on mRNAs that undergo subcellular localization prior to translation. In Saccharomyces cerevisiae, wild-type ASH1 mRNA is one of several dozen transcripts that are exported from the mother-cell nucleus during mitotic anaphase, transported to the bud tip on actin cables, anchored at the bud tip, and translated. Although repressed during transport, translation is a prerequisite for NMD. We found that ash1 nonsense mutations affect transport and/or anchoring independently of NMD. The nonsense transcripts respond to NMD in a manner dependent on the position of the mutation. Maximal sensitivity to NMD occurs when transport and translational repression are simultaneously impaired. Overall, our results suggest a model in which ash1 mRNAs are insensitive to NMD while translation is repressed during transport but become sensitive once repression is relieved.

Monitoring the temporal and spatial distribution of RNA in living yeast cells.

RNA localization is a cellular process to spatially restrict translation of specific proteins to defined regions within or between cells. Most localized mRNAs contain cis-acting localization elements in the 3'-untranslated region (UTR), which are sufficient for localization of an mRNA to a particular region of the cell. The cis-acting localization elements serve as assembly sites for trans-acting factors which function to sort the mRNA to the correct sub-cellular destination. Although fluorescent in situ hybridization (FISH) has been widely used to study mRNA localization, FISH has a weakness in that it is a static assay, as FISH requires that cells be fixed before hybridization. Consequently, FISH is not ideally suited for investigating dynamic mRNA localization processes. This limitation of FISH has been overcome by the development of techniques that allow the visualization of mRNA in living cells. Here, we present a protocol that tethers green fluorescent protein (GFP) to an mRNA of interest, allowing for the visualization of dynamic mRNA localization processes in living cells.

Loc1p is required for efficient assembly and nuclear export of the 60S ribosomal subunit.

Loc1p is an exclusively nuclear dsRNA-binding protein that affects the asymmetric sorting of ASH1 mRNA to daughter cells in Saccharomyces cerevisiae. In addition to the role in cytoplasmic RNA localization, Loc1p is a constituent of pre-60S ribosomes. Cells devoid of Loc1p display a defect in the synthesis of 60S ribosomal subunits, resulting in "half-mer" polyribosomes. Previously, we reported that Loc1p is located throughout the entire nucleus; however, upon closer inspection we discovered that Loc1p is enriched in the nucleolus consistent with a role in 60S ribosome biogenesis. Given that Loc1p is an RNA-binding protein and presumably functions in the assembly of 60S ribosomal subunits, we investigated if Loc1p has a role in rRNA processing and nuclear export of 60S subunits. Analysis of pre-rRNA processing revealed that loc1Delta cells exhibit gross defects in 25S rRNA synthesis, specifically a delay in processing at sites A0, A1 and A2 in 35S pre-rRNA. Furthermore, loc1Delta cells exhibit nuclear export defects for 60S ribosomal subunits, again, consistent with a role for Loc1p in the assembly of 60S ribosomal subunits. It is attractive to hypothesize that the two phenotypes associated with loc1Delta cells, namely altered ASH1 mRNA localization and ribosome biogenesis, are not mutually exclusive, but that ribosome biogenesis directly impacts mRNA localization.

Characterization of Mmp37p, a Saccharomyces cerevisiae mitochondrial matrix protein with a role in mitochondrial protein import.

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

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.

ASH1 mRNA anchoring requires reorganization of the Myo4p-She3p-She2p transport complex.

One mechanism by which cells post-transcriptionally regulate gene expression is via intercellular and intracellular sorting of mRNA. In Saccharomyces cerevisiae, the localization of ASH1 mRNA to the distal tip of budding cells results in the asymmetric sorting of Ash1p to daughter cell nuclei. Efficient localization of ASH1 mRNA depends upon the activity of four cis-acting localization elements and also upon the activity of trans-factors She2p, She3p, and Myo4p. She2p, She3p, and Myo4p have been proposed to form an ASH1 mRNA localization particle. She2p directly and specifically binds each of the four ASH1 cis-acting localization elements, whereas She3p has been hypothesized to function as an adaptor by recruiting the She2p-mRNA complex to Myo4p, a type V myosin. The Myo4p-She3p-She2p heterotrimeric protein complex has been proposed to localize mRNA to daughter cells using polarized actin cables. Here we demonstrate that whereas the predicted Myo4p-She3p-She2p heterotrimeric complex forms in vivo, it represents a relatively minor species compared with the Myo4p-She3p complex. Furthermore, contrary to a prediction of the heterotrimeric complex model for ASH1 mRNA localization, ASH1 mRNA artificially tethered to She2p is not localized. Upon closer examination, we found that mRNA tightly associated with She2p is transported to daughter cells but is not properly anchored at the bud tip. These results are consistent with a model whereby anchoring of ASH1 mRNA requires molecular remodeling of the Myo4p-She3p-She2p heterotrimeric complex, a process that is apparently altered when mRNA is artificially tethered to She2p.

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.

The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA(2) and cPLA(2) align or are similar to ExoU sequences. Site-specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.

mRNA decay: x (XRN1) marks the spot.

Degradation of mRNA is a vital aspect of gene expression. In yeast, Dcp1p, Dcp2p, Lsm1-7p, and Xrn1p are required for mRNA decay and are localized within discrete cytoplasmic foci; in the May 2 issue of Science, Sheth and Parker provide compelling evidence that these foci represent sites for mRNA decay.