The role of the 3' untranslated region in mRNA sorting to the vicinity of mitochondria is conserved from yeast to human cells

We recently demonstrated, using yeast DNA microarrays, that mRNAs of polysomes that coisolate with mitochondria code for a subset of mitochondrial proteins. The majority of these mRNAs encode proteins of prokaryotic origin. Herein, we show that a similar association occurs between polysomes and mitochondria in human cells. To determine whether mRNA transport machinery is conserved from yeast to human cells, we examined the subcellular localization of human OXA1 mRNA in yeast. Oxa1p is a key component in the biogenesis of mitochondrial inner membrane and is conserved from bacteria to eukaryotic organelles. The expression of human OXA1 cDNA partially restores the respiratory capacity of yeast oxa1- cells. In this study, we demonstrate that 1) OXA1 mRNAs are remarkably enriched in mitochondrion-bound polysomes purified from yeast and human cells; 2) the presence of the human OXA1 3' untranslated region (UTR) is required for the function of the human Oxa1p inside yeast mitochondria; and 3) the accurate sorting of the human OXA1 mRNA to the vicinity of yeast mitochondria is due to the recognition by yeast proteins of the human 3' UTR. Therefore, it seems that the recognition mechanism of OXA1 3' UTR is conserved throughout evolution and is necessary for Oxa1p function.     

Genome-wide analysis of mRNAs targeted to yeast mitochondria

It is agreed that nuclear-encoded mitochondrial proteins are post-translationally targeted to mitochondria, even if, in some cases, a co-translational phase can assist the import of precursor proteins. We used yeast DNA microarrays to analyse the mRNA populations associated with free and mitochondrion-bound polysomes. As expected, many mRNAs, known to encode mitochondrial proteins, are localized to free cytoplasmic polysomes, but many are localized to mitochondrion-bound polysomes. Furthermore, the 3′-UTR of six randomly chosen mitochondrion-bound mRNAs contains sufficient information to target, in vivo, non-translatable RNA to the vicinity of mitochondria. Interestingly, genes producing mRNAs that are targeted to mitochondria are mainly of ancient bacterial origin, whereas those producing mRNAs that are translated in the cytoplasm are mainly of eukaryotic origin. These observations, which support the recent hypotheses concerning the dual origin of the mitochondrial proteome, provide new insights into the biogenesis of mitochondria.     

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.     

Evidence that synthesis of the Saccharomyces cerevisiae mitochondrially encoded ribosomal protein Var1p may be membrane localized

The 5′-untranslated leaders of mitochondrial mRNAs appear to localize translation within the organelle. VAR1 is the only yeast mitochondrial gene encoding a major soluble protein. A chimeric mRNA bearing the VAR1 untranslated regions and the coding sequence for pre-Cox2p appears to be translated at the inner membrane surface. We propose that translation of the ribosomal protein Var1p is also likely to occur in close proximity to the inner membrane.     

Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae

Expression of yeast mitochondrial genes depends on specific translational activators acting on the 5'-untranslated region of their target mRNAs. Mss51p is a translational factor for cytochrome c oxidase subunit 1 (COX1) mRNA and a key player in down-regulating Cox1p expression when subunits with which it normally interacts are not available. Mss51p probably acts on the 5'-untranslated region of COX1 mRNA to initiate translation and on the coding sequence itself to facilitate elongation. Mss51p binds newly synthesized Cox1p, an interaction that could be necessary for translation. To gain insight into the different roles of Mss51p on Cox1p biogenesis, we have analyzed the properties of a new mitochondrial protein, mp15, which is synthesized in mss51 mutants and in cytochrome oxidase mutants in which Cox1p translation is suppressed. The mp15 polypeptide is not detected in cox14 mutants that express Cox1p normally. We show that mp15 is a truncated translation product of COX1 mRNA whose synthesis requires the COX1 mRNA-specific translational activator Pet309p. These results support a key role for Mss51p in translationally regulating Cox1p synthesis by the status of cytochrome oxidase assembly.     

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.     

Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs.

The essential products of the yeast mitochondrial translation system are seven hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small ribosomal subunit. Translation of the membrane proteins depends on nuclearly encoded, mRNA-specific translational activators that recognize the 5'-untranslated leaders of their target mRNAs. These translational activators are themselves membrane associated and could therefore tether translation to the inner membrane. In this study, we tested whether chimeric mRNAs with the untranslated sequences normally present on the mRNA encoding soluble Var1p, can direct functional expression of coding sequences specifying the integral membrane proteins Cox2p and Cox3p. DNA sequences specifying these chimeric mRNAs were inserted into mtDNA at the VAR1 locus and expressed in strains containing a nuclearly localized plasmid that supplies a functional form of Var1p, imported from the cytoplasm. Although cells expressing these chimeric mRNAs actively synthesized both membrane proteins, they were severely deficient in cytochrome c oxidase activity and in the accumulation of Cox2p and Cox3p, respectively. These data strongly support the physiological importance of interactions between membrane-bound mRNA-specific translational activators and the native 5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive synthesis of Cox2p and Cox3p to the inner membrane.     

The molecular basis for relative physiological functionality of the ADP/ATP carrier isoforms in Saccharomyces cerevisiae

AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.     

Coordination of endoplasmic reticulum and mRNA localization to the yeast bud

Localization of messenger RNAs and local protein synthesis contribute to asymmetric protein distribution not only of cytoplasmic but also of membrane or secreted proteins. Since synthesis of the latter protein classes occurs at the rough endoplasmic reticulum (ER), mRNA localization and distribution of ER should be coordinated. However, this coordination is not yet understood. In yeast, mRNA localization to the growing bud depends on the myosin Myo4p, its adaptor She3p, and the specific RNA binding protein She2p. These proteins mediate the localization of 23 mRNAs including ASH1 mRNA and mRNAs encoding membrane proteins. In addition, Myo4p and She3p are required for segregation of cortical ER to the bud. Here we show, with ASH1 mRNA as a model mRNA, that localizing messenger ribonucleoprotein (mRNP) particles comigrate with tubular ER structures to the bud, which requires the RNA binding protein She2p. Coordinated movement of the ASH1 mRNP with ER tubules but not their association with each other depends on Myo4p and She3p. Subcellular fractionation experiments demonstrate a cosegregation of ER and She2p, which is independent of Myo4p, She3p, or polysomes. Our findings suggest a novel model for mRNA localization that involves association of She2p and mRNPs with ER tubules and myosin-dependent cotransport of tubules and localized mRNPs.     

Import of proteins into mitochondria. Translatable mRNAs for imported mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes

In order to assess the role of different polysomal populations in mitochondrial protein import, yeast spheroplasts were treated with cycloheximide to prevent polysome "run-off" and fractionated into free polysomes, polysomes bound to the mitochondrial outer surface, and polysomes bound to the endoplasmic reticulum. These polysomes were analyzed for translatable mRNAs coding for 8 cytosolic and 12 imported mitochondrial proteins. The mitochondrial proteins included 7 proteins of the inner membrane, 2 proteins of the matrix, 2 proteins of the intermembrane space, and 1 protein of the outer membrane. Of the mRNAs for imported mitochondrial proteins, 8 were enriched in mitochondria-bound polysomes, 3 were enriched in free polysomes, and 1 was enriched in neither. All mRNAs for cytosolic proteins were enriched in free polysomes. Polysomes bound to the endoplasmic reticulum lacked significant levels of translatable mRNAs for either cytosolic or mitochondrial proteins. Even though mRNAs for imported mitochondrial proteins were enriched in mitochondria-bound polysomes, these polysomes represented only 12-18% of the total cytoplasmic polysomes. As a consequence, none of the translatable mRNAs for imported mitochondrial proteins tested was predominantly associated with mitochondria-bound polysomes. While mitochondria-bound polysomes may contribute to mitochondrial protein import, they do not appear to be obligatory for this process.     

The protein chaperone Ssa1 affects mRNA localization to the mitochondria

Many nuclear-transcribed mRNAs encoding mitochondrial proteins are localized near the mitochondrial outer membrane. A yet unresolved question is whether protein synthesis is important for transport of these mRNAs to their destination. Herein we present a connection between mRNA localization in yeast and the protein chaperone Ssa1. Ssa1 depletion lowered mRNA association with mitochondria while its overexpression increased it. A genome-wide analysis revealed that Ssa proteins preferentially affect mRNAs encoding hydrophobic proteins, which are expected targets for these protein chaperones. Importantly, deletion of the mitochondrial receptor Tom70 abolished the impact of Ssa1 overexpression on mRNAs encoding Tom70 targets. Taken together, our results suggest a role for Ssa1 in mediating localization of nascent peptide-ribosome-mRNA complexes to the mitochondria, consistent with a co-translational transport process.     

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.     

The 3' untranslated region of bovine follicle-stimulating hormone beta messenger RNA downregulates reporter expression: involvement of AU-rich elements and transfactors

FSHbeta mRNA has a unique 3' untranslated region (3'UTR) that is highly conserved across the species. Sequence analyses of the mouse, rat, human, bovine, and ovine 3'UTRs revealed the presence of elements implicated in mRNA instability and translational control such as AU-Rich Element (ARE) and lipoxygenase differentiation control elements. Bovine FSHbeta 3'UTR down-regulated reporter expression in alphaT3-1 and NIH3T3 cells, but not in HEK 293 cells, suggesting the involvement of a cell-specific factor or mechanism. The presence of a 3'UTR did not influence reporter mRNA stability, but it did decrease its association with polysomes, indicating that the downregulatory effect may be exerted at the translational level. The segment spanning 601-800 bases (U4) of the bovine FSHbeta 3'UTR was found to be the most effective downregulating segment, its effect being equal to that of the full-length 3'UTR. RNA electrophoretic mobility shift assay with U4 showed the presence of specific transfactors in the cytosolic preparations of bovine pituitary and the cell lines. U4 contained an ARE that appeared to be functional, because the mutated U4 ARE was ineffective in downregulating the reporter expression and inhibiting [(32)P]-labeled U4-transfactor complex formation. Downregulation of reporter activity by the full-length 3'UTR and U4 could be overcome by overexpression of HuR, a protein known to stabilize ARE-containing mRNAs in NIH3T3 cells, but not in the alphaT3-1 cells, once again indicating that factors other than HuR may also be involved in the regulation of FSHbeta in the pituitary.