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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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Validation of an in vivo selection for mRNA mitochondrial localization (17). Plasmid pATP2 expressing either a negative control 3′ UTR (pATP2c) or the wild-type ATP2 3′ UTR (pATP2-636) was introduced into strain ATP2-3′ADH1. Ten-fold serial dilutions of the resulting strains were plated on media (-trp -ura) under either nonselective conditions (with glucose as the sole carbon source, top) or selective conditions (with glycerol as the sole carbon source, bottom).
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Figure 1: Validation of an in vivo selection for mRNA mitochondrial localization (17). Plasmid pATP2 expressing either a negative control 3′ UTR (pATP2c) or the wild-type ATP2 3′ UTR (pATP2-636) was introduced into strain ATP2-3′ADH1. Ten-fold serial dilutions of the resulting strains were plated on media (-trp -ura) under either nonselective conditions (with glucose as the sole carbon source, top) or selective conditions (with glycerol as the sole carbon source, bottom).

Mentions: This selection method was validated by introducing into ATP2-3′ADH1 cells plasmid pATP2 expressing either the full-length ATP2 3′ UTR (pATP2–636), an active, shortened ATP2 3′ UTR (first 250 nt; pATP2-250) (17) or a random 500 nt 3′ UTR (pATP2c). The full-length 3′ UTR from ATM1, another nuclear-encoded mitochondrial protein, was used as an additional positive control (pATP2-ATM1) (26). When plated on synthetic medium containing glycerol, a survival rate of only 0.05% was observed in cells carrying the random 3′ UTR plasmid, pATP2c (Figure 1). In contrast, when either of the two ATP2 3′ UTR variants (636 or 250 nt) or the ATM1 3′ UTR was expressed, >50% of the cells plated survived on glycerol medium (Figure 1 and data not shown). These results suggest that the above selection successfully links cell survival with having localization-competent 3′ UTRs in a manner that does not rely on ATP2-specific 3′ UTR elements.Figure 1.


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)

Validation of an in vivo selection for mRNA mitochondrial localization (17). Plasmid pATP2 expressing either a negative control 3′ UTR (pATP2c) or the wild-type ATP2 3′ UTR (pATP2-636) was introduced into strain ATP2-3′ADH1. Ten-fold serial dilutions of the resulting strains were plated on media (-trp -ura) under either nonselective conditions (with glucose as the sole carbon source, top) or selective conditions (with glycerol as the sole carbon source, bottom).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175308&req=5

Figure 1: Validation of an in vivo selection for mRNA mitochondrial localization (17). Plasmid pATP2 expressing either a negative control 3′ UTR (pATP2c) or the wild-type ATP2 3′ UTR (pATP2-636) was introduced into strain ATP2-3′ADH1. Ten-fold serial dilutions of the resulting strains were plated on media (-trp -ura) under either nonselective conditions (with glucose as the sole carbon source, top) or selective conditions (with glycerol as the sole carbon source, bottom).
Mentions: This selection method was validated by introducing into ATP2-3′ADH1 cells plasmid pATP2 expressing either the full-length ATP2 3′ UTR (pATP2–636), an active, shortened ATP2 3′ UTR (first 250 nt; pATP2-250) (17) or a random 500 nt 3′ UTR (pATP2c). The full-length 3′ UTR from ATM1, another nuclear-encoded mitochondrial protein, was used as an additional positive control (pATP2-ATM1) (26). When plated on synthetic medium containing glycerol, a survival rate of only 0.05% was observed in cells carrying the random 3′ UTR plasmid, pATP2c (Figure 1). In contrast, when either of the two ATP2 3′ UTR variants (636 or 250 nt) or the ATM1 3′ UTR was expressed, >50% of the cells plated survived on glycerol medium (Figure 1 and data not shown). These results suggest that the above selection successfully links cell survival with having localization-competent 3′ UTRs in a manner that does not rely on ATP2-specific 3′ UTR elements.Figure 1.

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
Related in: MedlinePlus