Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.     

A genomic integration method to visualize localization of endogenous mRNAs in living yeast

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).     

mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface-the region coding for the mitochondrial targeting sequence and the 3'UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3'UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3'UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity.     

Localization of a Subset of Yeast mRNAs Depends on Inheritance of Endoplasmic Reticulum

Localization of messenger RNA (mRNAs) contributes to generation and maintenance of cellular asymmetry, embryonic development and neuronal function. The She1‐3 protein machinery in Saccharomyces cerevisiae localizes >30 mRNAs to the bud tip, including 13 mRNAs encoding membrane or secreted proteins. Ribonucleoprotein (RNP) particles can co‐localize with tubular endoplasmic reticulum (ER) structures that form the initial elements for segregation of cortical ER (cER), suggesting a coordination of mRNA localization and cER distribution. By investigating localization of MS2‐tagged mRNAs in yeast defective at various stages of cER segregation, we demonstrate that proper cER segregation is required for localization of only a subset of mRNAs. These mRNAs include WSC2, IST2, EAR1 and SRL1 that encode membrane or ER associated proteins and are expressed during S and G2 phases of the cell cycle when tubular ER movement into the bud occurs. Translation of WSC2 is not required for localization, ruling out co‐translational targeting of this mRNA. Localization of ASH1 mRNA is independent of cER segregation, which is consistent with the expression pattern of ASH1 at late mitosis. Our findings indicate the presence of two different pathways to localize mRNAs to the yeast bud.     

In yeast, the 3' untranslated region or the presequence of ATM1 is required for the exclusive localization of its mRNA to the vicinity of mitochondria

We isolated mitochondria from Saccharomyces cerevisiae to selectively study polysomes bound to the mitochondrial surface. The distribution of several mRNAs coding for mitochondrial proteins was examined in free and mitochondrion-bound polysomes. Some mRNAs exclusively localize to mitochondrion-bound polysomes, such as the ones coding for Atm1p, Cox10p, Tim44p, Atp2p, and Cot1p. In contrast, mRNAs encoding Cox6p, Cox5a, Aac1p, and Mir1p are found enriched in free cytoplasmic polysome fractions. Aac1p and Mir1p are transporters that lack cleavable presequences. Sequences required for mRNA asymmetric subcellular distribution were determined by analyzing the localization of reporter mRNAs containing the presequence coding region and/or the 3'-untranslated region (3'UTR) of ATM1, a gene encoding an ABC transporter of the mitochondrial inner membrane. Biochemical analyses of mitochondrion-bound polysomes and direct visualization of RNA localization in living yeast cells allowed us to demonstrate that either the presequence coding region or the 3'UTR of ATM1 is sufficient to allow the reporter mRNA to localize to the vicinity of the mitochondrion, independently of its translation. These data demonstrate that mRNA localization is one of the mechanisms used, in yeast, for segregating mitochondrial proteins.     

Determinants of mRNA localization

RNA localization provides a mechanism for protein targeting within developing or differentiating cells. Specific cis-acting sequences on mRNA mediate this process. Such 'localizer' or 'zipcode' nucleic acid sequences have been restricted to the 3' untranslated region of several mRNAs. The presence of genetic information denoting a spatial component of translation adds a new dimension to gene expression.     

Evidence that the GCN2 protein kinase regulates reinitiation by yeast ribosomes.

The yeast gene GCN4 produces an mRNA that has a long 5' 'untranslated' region containing four small open reading frames (ORFs) preceding the protein coding frame. This configuration suppresses the rate by which GCN4 protein is synthesized. However, translational derepression of the GCN4 mRNA occurs when yeast cells are grown under conditions of amino acid limitation. Such translational derepression requires the GCN2 protein kinase and the presence of the 5' most proximal ORF. In this study we show that a functional coupling between the translation of the first ORF and the amount of the GCN2 protein is responsible for the translational derepression of the GCN4 mRNA. Our evidence suggests that this coupling involves an increase in the ability of 40S ribosomal subunits that have translated the first frame to resume scanning and reinitiate translation at a downstream AUG independently of the base sequence in the intervening region.     

Proteins binding to 5'' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.     

mRNAs encoding polarity and exocytosis factors are cotransported with the cortical endoplasmic reticulum to the incipient bud in Saccharomyces cerevisiae

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 (myo4Delta, sec3Delta, 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.     

Can ID repetitive elements serve as cis-acting dendritic targeting elements? An in vivo study

Dendritic localization of mRNA/RNA involves interaction of cis-elements and trans-factors. Small, non-protein coding dendritic BC1 RNA is thought to regulate translation in dendritic microdomains. Following microinjections into cultured cells, BC1 RNA fused to larger mRNAs appeared to impart transport competence to these chimeras, and its 5' ID region was proposed as the cis-acting dendritic targeting element. As these ID elements move around rodent genomes and, if transcribed, form a long RNA stem-loop, they might, thereby, lead to new localizations for targeted gene products. To test their targeting ability in vivo we created transgenic mice expressing various ID elements fused to the 3' UTR of reporter mRNA for Enhanced Green Fluorescent Protein. In vivo, neither ID elements nor the BC1 RNA coding region were capable of transporting EGFP RNA to dendrites, although the 3' UTR of alpha-CaMKII mRNA, an established cis-acting element did produce positive results. Other mRNAs containing naturally inserted ID elements are also not found in neuronal dendrites. We conclude that the 5' ID domain from BC1 RNA is not a sufficient dendritic targeting element for mRNAs in vivo.     

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.     

Simultaneous transport of different localized mRNA species revealed by live-cell imaging

Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.