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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 NRR-diversified ATP2 3′ UTR variants before selection. The numbering across the top of the figure corresponds to the nucleotide position in the 3′ UTR of ATP2, where ‘1’ is the first nucleotide immediately after the stop codon. Each arrow represents an ATP2 fragment. Arrow positions indicate the origin of each fragment within the parental ATP2 gene. Arrow colors indicate the order of the fragment reassembly (5′-red-orange-yellow … black-3′, as shown at the bottom of the figure). The direction of each arrow identifies whether the fragment came from the sense (pointing right) or antisense (pointing left) strand of ATP2. The clone names beginning with ‘U1’, ‘U2’ and ‘U3’ came from library L1, L2 and L3, respectively.
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Figure 2: Composition of NRR-diversified ATP2 3′ UTR variants before selection. The numbering across the top of the figure corresponds to the nucleotide position in the 3′ UTR of ATP2, where ‘1’ is the first nucleotide immediately after the stop codon. Each arrow represents an ATP2 fragment. Arrow positions indicate the origin of each fragment within the parental ATP2 gene. Arrow colors indicate the order of the fragment reassembly (5′-red-orange-yellow … black-3′, as shown at the bottom of the figure). The direction of each arrow identifies whether the fragment came from the sense (pointing right) or antisense (pointing left) strand of ATP2. The clone names beginning with ‘U1’, ‘U2’ and ‘U3’ came from library L1, L2 and L3, respectively.

Mentions: To assess the diversity introduced by NRR, 19 library members from L1 to L3 were characterized by DNA sequencing prior to any functional selection (Figure 2). The composition of the diversified library members was observed to be consistent with the library design parameters. For example, the characterized sequences from L1 averaged 215 bp in length and contained up to five crossovers, between fragments ranging in size from 14 to 300 nt. Similarly, the characterized sequences from L3 averaged 100 bp in length and contained up to four crossovers, between fragments ranging in size from 18 to 54 bp.Figure 2.


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 NRR-diversified ATP2 3′ UTR variants before selection. The numbering across the top of the figure corresponds to the nucleotide position in the 3′ UTR of ATP2, where ‘1’ is the first nucleotide immediately after the stop codon. Each arrow represents an ATP2 fragment. Arrow positions indicate the origin of each fragment within the parental ATP2 gene. Arrow colors indicate the order of the fragment reassembly (5′-red-orange-yellow … black-3′, as shown at the bottom of the figure). The direction of each arrow identifies whether the fragment came from the sense (pointing right) or antisense (pointing left) strand of ATP2. The clone names beginning with ‘U1’, ‘U2’ and ‘U3’ came from library L1, L2 and L3, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Composition of NRR-diversified ATP2 3′ UTR variants before selection. The numbering across the top of the figure corresponds to the nucleotide position in the 3′ UTR of ATP2, where ‘1’ is the first nucleotide immediately after the stop codon. Each arrow represents an ATP2 fragment. Arrow positions indicate the origin of each fragment within the parental ATP2 gene. Arrow colors indicate the order of the fragment reassembly (5′-red-orange-yellow … black-3′, as shown at the bottom of the figure). The direction of each arrow identifies whether the fragment came from the sense (pointing right) or antisense (pointing left) strand of ATP2. The clone names beginning with ‘U1’, ‘U2’ and ‘U3’ came from library L1, L2 and L3, respectively.
Mentions: To assess the diversity introduced by NRR, 19 library members from L1 to L3 were characterized by DNA sequencing prior to any functional selection (Figure 2). The composition of the diversified library members was observed to be consistent with the library design parameters. For example, the characterized sequences from L1 averaged 215 bp in length and contained up to five crossovers, between fragments ranging in size from 14 to 300 nt. Similarly, the characterized sequences from L3 averaged 100 bp in length and contained up to four crossovers, between fragments ranging in size from 18 to 54 bp.Figure 2.

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