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Discovery of a mRNA mitochondrial localization element in Saccharomyces cerevisiae by nonhomologous random recombination and in vivo selection.

Liu JM, Liu DR - Nucleic Acids Res. (2007)

Bottom Line: To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3' UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization.Site-directed mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity.In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 01238, USA.

ABSTRACT
In budding yeast, over 100 nuclear-encoded mRNAs are localized to the mitochondria. The determinants of mRNA localization to the mitochondria are not well understood, and protein factors involved in this process have not yet been identified. To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3' UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization. When applied to the yeast ATP2 mRNA, this approach rapidly identified a 50-nt consensus motif, designated Min2, as well as two Min2-homologous regions naturally present downstream of the ATP2 stop codon, which are sufficient when appended to the 3' end of various reporter mRNAs to induce mitochondrial localization. Site-directed mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity. In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.

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Composition of 3′ UTR variants surviving selection for mitochondrial localization. Sequences shown are able to localize ATP2 mRNA to the mitochondria, as determined by their ability to rescue ATP2-3′ADH1 cell growth on glycerol media when expressed from pATP2. The labeling scheme is as described in Figure 2. The 14 variants highlighted in green and red were also screened with the green-RNA system; the 13 variants boxed in green were able to localize GFP-labeled ATP2 mRNA to the mitochondria, while 4–53 did not localize gRNA-particles to the mitochondria (see text and Figure 4). Vertical black lines indicate fragments that run through one of the two regions of the ATP2 3′ UTR (nt 263–312 and nt 164–213) that share homology with minimal localization motif Min2.
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Figure 3: Composition of 3′ UTR variants surviving selection for mitochondrial localization. Sequences shown are able to localize ATP2 mRNA to the mitochondria, as determined by their ability to rescue ATP2-3′ADH1 cell growth on glycerol media when expressed from pATP2. The labeling scheme is as described in Figure 2. The 14 variants highlighted in green and red were also screened with the green-RNA system; the 13 variants boxed in green were able to localize GFP-labeled ATP2 mRNA to the mitochondria, while 4–53 did not localize gRNA-particles to the mitochondria (see text and Figure 4). Vertical black lines indicate fragments that run through one of the two regions of the ATP2 3′ UTR (nt 263–312 and nt 164–213) that share homology with minimal localization motif Min2.

Mentions: The three NRR-diversified 3′ UTR libraries were each introduced into yeast cells, and the resulting transformants were selected for their ability to localize the ATP2 mRNA to the mitochondria as described above. For each of the three NRR-based libraries, we observed that 1 in ∼250 transformants (from 105 total transformants) were able to survive under selective conditions, regardless of the library construction method. A subset of surviving colonies was further characterized by subcloning their UTR inserts into fresh pATP2, followed by reselection to confirm their activity. In total, 45 clones from L1 to L3 were characterized as exhibiting growth phenotypes under selective conditions consistent with mitochondrial localization of ATP2 mRNA (Figure 3). Among these 45 sequences, 14 were characterized by fluorescence microscopy using the green-RNA system, of which 13 (92%) were observed to colocalize with MitoTracker (Figure 4A; clone 4–53 failed to colocalize with MT, data not shown). The high degree of correspondence between the selection phenotypes and the corresponding green-RNA observations is consistent with the basic assumption that the selection system enriches library members with mitochondrial localization activity.Figure 3.


Discovery of a mRNA mitochondrial localization element in Saccharomyces cerevisiae by nonhomologous random recombination and in vivo selection.

Liu JM, Liu DR - Nucleic Acids Res. (2007)

Composition of 3′ UTR variants surviving selection for mitochondrial localization. Sequences shown are able to localize ATP2 mRNA to the mitochondria, as determined by their ability to rescue ATP2-3′ADH1 cell growth on glycerol media when expressed from pATP2. The labeling scheme is as described in Figure 2. The 14 variants highlighted in green and red were also screened with the green-RNA system; the 13 variants boxed in green were able to localize GFP-labeled ATP2 mRNA to the mitochondria, while 4–53 did not localize gRNA-particles to the mitochondria (see text and Figure 4). Vertical black lines indicate fragments that run through one of the two regions of the ATP2 3′ UTR (nt 263–312 and nt 164–213) that share homology with minimal localization motif Min2.
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Related In: Results  -  Collection

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Figure 3: Composition of 3′ UTR variants surviving selection for mitochondrial localization. Sequences shown are able to localize ATP2 mRNA to the mitochondria, as determined by their ability to rescue ATP2-3′ADH1 cell growth on glycerol media when expressed from pATP2. The labeling scheme is as described in Figure 2. The 14 variants highlighted in green and red were also screened with the green-RNA system; the 13 variants boxed in green were able to localize GFP-labeled ATP2 mRNA to the mitochondria, while 4–53 did not localize gRNA-particles to the mitochondria (see text and Figure 4). Vertical black lines indicate fragments that run through one of the two regions of the ATP2 3′ UTR (nt 263–312 and nt 164–213) that share homology with minimal localization motif Min2.
Mentions: The three NRR-diversified 3′ UTR libraries were each introduced into yeast cells, and the resulting transformants were selected for their ability to localize the ATP2 mRNA to the mitochondria as described above. For each of the three NRR-based libraries, we observed that 1 in ∼250 transformants (from 105 total transformants) were able to survive under selective conditions, regardless of the library construction method. A subset of surviving colonies was further characterized by subcloning their UTR inserts into fresh pATP2, followed by reselection to confirm their activity. In total, 45 clones from L1 to L3 were characterized as exhibiting growth phenotypes under selective conditions consistent with mitochondrial localization of ATP2 mRNA (Figure 3). Among these 45 sequences, 14 were characterized by fluorescence microscopy using the green-RNA system, of which 13 (92%) were observed to colocalize with MitoTracker (Figure 4A; clone 4–53 failed to colocalize with MT, data not shown). The high degree of correspondence between the selection phenotypes and the corresponding green-RNA observations is consistent with the basic assumption that the selection system enriches library members with mitochondrial localization activity.Figure 3.

Bottom Line: To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3' UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization.Site-directed mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity.In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 01238, USA.

ABSTRACT
In budding yeast, over 100 nuclear-encoded mRNAs are localized to the mitochondria. The determinants of mRNA localization to the mitochondria are not well understood, and protein factors involved in this process have not yet been identified. To reveal the sequence determinants for mitochondrial localization in a comprehensive and unbiased manner, we generated highly diversified libraries of 3' UTR regions from a known mitochondrially localized mRNA by nonhomologous random recombination (NRR) and subjected the resulting sequences to an in vivo selection that links cell survival to mitochondrial mRNA localization. When applied to the yeast ATP2 mRNA, this approach rapidly identified a 50-nt consensus motif, designated Min2, as well as two Min2-homologous regions naturally present downstream of the ATP2 stop codon, which are sufficient when appended to the 3' end of various reporter mRNAs to induce mitochondrial localization. Site-directed mutagenesis of Min2 revealed primary and secondary structure elements that contribute to localization activity. In addition, the Min2 motif may facilitate the identification of proteins involved in this mode of establishing cellular asymmetry.

Show MeSH