Analysis of the Saccharomyces cerevisiae mitochondrial COX3 mRNA 5' untranslated leader: translational activation and mRNA processing.

We used transformation of yeast mitochondria and homologous gene replacement to study features of the 613-base COX3 mRNA 5' untranslated leader (5'-UTL) required for translational activation by the protein products of the nuclear genes PET54, PET122, and PET494 in vivo. Elimination of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowing ribosomes to bypass that AUG. Deletion of the entire 5'-UTL completely prevented translation, suggesting that the activator proteins do not function by antagonizing any other negative element in the 5'-UTL. Removal of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator-dependent translation. The largest internal deletion that did not interfere with translation removed 125 bases from the upstream portion of the leader. However, two large deletions that blocked translation could be reverted to activator-dependent expression by secondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it. Taken together, the deletion mutations and revertants define a region of 151 bases (between positions -480 and -330 relative to the start codon) containing sequences that are sufficient for translational activation when modified slightly. Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PET54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins. The mature COX3 mRNA is cleaved from a precursor immediately downstream of the preceding tRNAVal in a fashion resembling mRNA processing in vertebrate mitochondria. Our results indicate that the site of this cleavage in Saccharomyces cerevisiae is determined solely by the position of the tRNA.     

Role for Upf2p phosphorylation in Saccharomyces cerevisiae nonsense-mediated mRNA decay

Premature termination (nonsense) codons trigger rapid mRNA decay by the nonsense-mediated mRNA decay (NMD) pathway. Two conserved proteins essential for NMD, UPF1 and UPF2, are phosphorylated in higher eukaryotes. The phosphorylation and dephosphorylation of UPF1 appear to be crucial for NMD, as blockade of either event in Caenorhabditis elegans and mammals largely prevents NMD. The universality of this phosphorylation/dephosphorylation cycle pathway has been questioned, however, because the well-studied Saccharomyces cerevisiae NMD pathway has not been shown to be regulated by phosphorylation. Here, we used in vitro and in vivo biochemical techniques to show that both S. cerevisiae Upf1p and Upf2p are phosphoproteins. We provide evidence that the phosphorylation of the N-terminal region of Upf2p is crucial for its interaction with Hrp1p, an RNA-binding protein that we previously showed is essential for NMD. We identify specific amino acids in Upf2p's N-terminal domain, including phosphorylated serines, which dictate both its interaction with Hrp1p and its ability to elicit NMD. Our results indicate that phosphorylation of UPF1 and UPF2 is a conserved event in eukaryotes and for the first time provide evidence that Upf2p phosphorylation is crucial for NMD.     

Alteration of the Saccharomyces cerevisiae COX2 mRNA 5'-untranslated leader by mitochondrial gene replacement and functional interaction with the translational activator protein PET111.

The ability to replace wild-type mitochondrial DNA sequences in yeast with in vitro-generated mutations has been exploited to study the mechanism by which the nuclearly encoded PET111 protein specifically activates translation of the mitochondrially coded COX2 mRNA. We have generated three mutations in vitro that alter the COX2 mRNA 5'-untranslated leader (UTL) and introduced them into the mitochondrial genome, replacing the wild-type sequence. None of the mutations significantly affected the steady-state level of COX2 mRNA. Deletion of a single base at position -24 (relative to the translation initiation codon) in the 5'-UTL (cox2-11) reduced COX2 mRNA translation and respiratory growth, whereas insertion of four bases in place of the deleted base (cox2-12) and deletion of bases -30 to -2 (cox2-13) completely blocked both. Six spontaneous nuclear mutations were selected as suppressors of the single-base 5'-UTL deletion, cox2-11. One of these mapped to PET111 and was shown to be a missense mutation that changed residue 652 from Ala to Thr. This suppressor, PET111-20, failed to suppress the 29-base deletion, cox2-13, but very weakly suppressed the insertion mutation, cox2-12. PET111-20 also enhanced translation of a partially functional COX2 mRNA with a wild-type 5'-UTL but a mutant initiation codon. Although overexpression of the wild-type PET111 protein caused weak suppression of the single-base deletion, cox2-11, the PET111-20 suppressor mutation did not function simply by increasing the level of the protein. These results demonstrate an intimate functional interaction between the translational activator protein and the mRNA 5'-UTL and suggest that they may interact directly.     

CA repeats in the 3'-untranslated region of bcl-2 mRNA mediate constitutive decay of bcl-2 mRNA

An AU-rich element (ARE) in the 3'-untranslated region (UTR) of bcl-2 mRNA has previously been shown to be responsible for destabilizing bcl-2 mRNA during apoptosis through increasing AUF1 binding. In the present study, we investigated the effect of the region upstream of the ARE on bcl-2 mRNA stability using serial deletion constructs of the 3'-UTR of bcl-2. Deletion of 30 nucleotides mostly consisting of the CA repeats, located upstream of the ARE, resulted in the stabilization of bcl-2 mRNA abundance, in the absence or presence of the ARE. The specificity of the CA repeats in terms of destabilizing bcl-2 mRNA was proven by the substituting the CA repeats with other alternative repeats of purine/pyrimidine, but this had no effect on the stability of bcl-2 mRNA. CA repeats alone, however, failed to confer instability to bcl-2 or gfp reporter mRNAs, indicating a requirement for additional sequences in the upstream region of the 3'-UTR. Serial deletion and replacement of a part of the region upstream of the CA repeats revealed that the entire 131-nucleotide upstream region is an essential prerequisite for the CA repeat-dependent destabilization of bcl-2 mRNA. Unlike the ARE, CA repeat-mediated degradation of bcl-2 mRNA was not accelerated upon apoptotic stimulus. Moreover, the upstream sequences and CA repeats are conserved among mammals. Collectively, CA repeats contribute to the constitutive decay of bcl-2 mRNA in the steady states, thereby maintaining appropriate bcl-2 levels in mammalian cells.     

Genetic evidence for interaction between Cbp1 and specific nucleotides in the 5' untranslated region of mitochondrial cytochrome b mRNA in Saccharomyces cerevisiae.

The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors. The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA. A previous deletion analysis identified an 11-nucleotide-long sequence within the 5' untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability. In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element. The CCG sequence within this region was shown to be necessary for stability. A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element. This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation.     

Multiple splice forms of ribonuclease-inhibitor mRNA differ in the 5'-untranslated region

We report the sequence of the cDNAs representing five independent splice forms of human placental RNase inhibitor (RI) mRNA. RI mRNAs differ principally in the 5'-untranslated region, which may include or lack a 68-nucleotide (nt) exon inserted at a splice site located only 20 nt upstream from the initiator AUG. At least three other exons may also abut the same splice site. This unusual and variable feature of the mRNA would suggest that secondary structure in the region of the start codon may differ among RI messages. A single copy of the RI gene exists in the human genome.     

Genetic analysis of complex interactions among components of the mitochondrial import motor and translocon in Saccharomyces cerevisiae

A highly conserved, Hsp70-based, import motor, which is associated with the translocase on the matrix side of the inner mitochondrial membrane, is critical for protein translocation into the matrix. Hsp70 is tethered to the translocon via interaction with Tim44. Pam18, the J-protein co-chaperone, and Pam16, a structurally related protein with which Pam18 forms a heterodimer, are also critical components of the motor. Their N termini are important for the heterodimer's translocon association, with Pam18's and Pam16's N termini interacting in the intermembrane space and the matrix, respectively. Here, using the model organism Saccharomyces cerevisiae, we report the identification of an N-terminal segment of Tim44, important for association of Pam16 with the translocon. We also report that higher amounts of Pam17, a nonessential motor component, are found associated with the translocon in both PAM16 and TIM44 mutants that affect their interaction with one another. These TIM44 and PAM16 mutations are also synthetically lethal with a deletion of PAM17. In contrast, a deletion of PAM17 has little, or no genetic interaction with a PAM18 mutation that affects translocon association of the Pam16:Pam18 heterodimer, suggesting a second role for the Pam16:Tim44 interaction. A similar pattern of genetic interactions and enhanced Pam17 translocon association was observed in the absence of the C terminus of Tim17, a core component of the translocon. We suggest the Pam16:Tim44 interaction may play two roles: (1) tethering the Pam16:Pam18 heterodimer to the translocon and (2) positioning the import motor for efficient engagement with the translocating polypeptide along with Tim17 and Pam17.     

Suppressor analysis of the mpt5/htr1/uth4/puf5 deletion in Saccharomyces cerevisiae

The MPT5/HTR1/UTH4/PUF5 gene encodes an RNA-binding Puf-family protein in Saccharomyces cerevisiae. The Δmpt5 cells exhibit pleiotropic phenotypes, including the G2/M arrest of the cell cycle and weakened cell wall at high temperatures. The Δmpt5 disruptant was also hydroxyurea (HU) sensitive. In this study we screened deletion suppressors to rescue the temperature sensitivity of Δmpt5, and identified dsf1 (YEL070W), dsf2 (YBR007C), sir2, sir3, sir4 and swe1. Multicopy suppressors identified were PKC1 and its upstream genes, but not the downstream MAPK cascade genes. The overexpression of PKC1, however, did not suppress the HU sensitivity of Δmpt5. In contrast, both the HU- and temperature-sensitivities of a-type Δmpt5 cells were suppressed by each sir deletion or a multicopy of MATα2, suggesting that a diploid-type expression is involved. We found that a diploid-specific IME4 gene encoding an RNA-modifying protein was responsible for the suppression of the temperature sensitivity, but not of the HU sensitivity. Furthermore, the suppression of the HU sensitivity depended on PUF4, another Puf-family gene, and overexpression of PUF4 suppressed only the HU sensitivity of Δmpt5. The protein level of Puf4 was not affected by the sir mutation. Thus, these Ime4 and Puf4 proteins play complementary roles to rescue the defects in Δmpt5 Δsir cells.     

Genome-wide analysis of mRNA lengths in Saccharomyces cerevisiae

Background  

Although the protein-coding sequences in the Saccharomyces cerevisiae genome have been studied and annotated extensively, much less is known about the extent and characteristics of the untranslated regions of yeast mRNAs.     

Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine and cardiolipin biosynthetic pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae mitochondria contain enzymes required for synthesis of the phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE), which are enriched in mitochondrial membranes. Previous studies indicated that PE may compensate for the lack of CL, and vice versa. These data suggest that PE and CL have overlapping functions and that the absence of both lipids may be lethal. To address this hypothesis, we determined whether the crd1delta mutant, which lacks CL, was viable in genetic backgrounds in which PE synthesis was genetically blocked. Deletion of the mitochondrial PE pathway gene PSD1 was synthetically lethal with the crd1delta mutant, whereas deletion of the Golgi and endoplasmic reticulum pathway genes PSD2 and DPL1 did not result in synthetic lethality. A 20-fold reduction in phosphatidylcholine did not affect the growth of crd1delta cells. Supplementation with ethanolamine, which led to increased PE synthesis, or with propanolamine, which led to synthesis of the novel phospholipid phosphatidylpropanolamine, failed to rescue the synthetic lethality of the crd1delta psd1delta cells. These results suggest that mitochondrial biosynthesis of PE is essential for the viability of yeast mutants lacking CL.