Roy M. Long

Spatial regulation of translation through RNA localization

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. WIREs RNA 2011 2 601–609 DOI: 10.1002/wrna.83 For further resources related to this article, please visit the WIREs website

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.

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.

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 loc1Δ cells exhibit gross defects in 25S rRNA synthesis, specifically a delay in processing at sites A0, A1 and A2 in 35S pre-rRNA. Furthermore, loc1Δ 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 loc1Δ cells, namely altered ASH1 mRNA localization and ribosome biogenesis, are not mutually exclusive, but that ribosome biogenesis directly impacts mRNA localization.

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.

InSaccharomyces cerevisiae,ASH1mRNA is localized to the tip of daughter cells during anaphase of the cell cycle.ASH1mRNA localization is dependent on fourcis-acting localization elements as well as Myo4p, She2p, and She3p. Myo4p, She2p, and She3p are hypothesized to form a heterotrimeric protein complex that directly transportsASH1mRNA to daughter cells. She2p is an RNA-binding protein that directly interacts withASH1 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 ofASH1mRNA. 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.

▪ Abstract The localization of mRNAs is used by various types of polarized cells to locally translate specific proteins, which restricts their distribution to a particular sub-region of the cytoplasm. This mechanism of protein sorting is involved in major biological processes such as asymmetric cell division, oogenesis, cellular motility, and synapse formation. With the finding of localized mRNAs in the yeast Saccharomyces cerevisiae, it is now possible to benefit from the powerful yeast laboratory tools to explore the molecular basis of RNA localization. Because mRNA transport and localization in yeast share many features with RNA localization in higher eukaryotes, including the formation of a large ribonucleoprotein (RNP) localization complex, the requirement of a polarized cytoskeleton and molecular motors, and the role of nuclear RNA-binding proteins in cytoplasmic localization, the yeast can be used as a paradigm for unraveling the molecular aspects of this process. This review summarizes the current knowledge on RNP transport and localization 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.

In Saccharomyces cerevisiae , Ash1p is a specific repressor of transcription that localizes exclusively to daughter cell nuclei through the asymmetric localization of ASH1 mRNA. This localization requires four cis ‐acting localization elements located in the ASH1 mRNA, five trans ‐acting factors, one of which is a myosin, and the actin cytoskeleton. The RNA‐binding proteins that interact with these cis ‐elements remained to be identified. Starting with the 3′ most localization element of ASH1 mRNA in the three‐hybrid assay, element E3, we isolated a clone corresponding to the C‐terminus of She3p. We also found that She3p and She2p interact, and this interaction is essential for the binding of She3p with element E3 in vivo . Moreover, She2p was observed to bind the E3 RNA directly in vitro and each of the ASH1 cis ‐acting localization elements requires She2p for their localization function. By tethering a She3p–MS2 fusion protein to a reporter RNA containing MS2 binding sites, we observed that She2p is dispensable for She3p–MS2‐dependent RNA localization.