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Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.

Truong DM, Hewitt FC, Hanson JH, Cui X, Lambowitz AM - PLoS Genet. (2015)

Bottom Line: Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells.By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+.Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

No MeSH data available.


Related in: MedlinePlus

Mutational fitness map of the Ll.LtrB intron during directed evolution in human cells.Secondary structure diagram of the Ll.LtrB intron showing mutation frequencies based on Pacific Biosciences RS circular consensus sequencing after 8 and 12 cycles of directed evolution in HEK-293 cells in culture medium supplemented with 80 mM or 40 mM MgCl2. Round 8 was assessed with 1,395 full-length reads, with the results presented as a color-coded heat map on a secondary structure diagram of the Ll.LtrB intron. Dark to light blue ovals represent conserved nucleotide positions with mutations present in 0–0.3% of the population. Pink to red ovals indicate mutable nucleotide positions with mutations present in >0.3–60% of the population. Green triangles with nucleotide inscribed, indicate positive selection in rounds 1–8 with the indicated nucleotide comprising >80% of the mutations at that position. Round 12 was assessed with 3,069 full-length reads with large colored arrows indicating positions at which the indicated mutation increased (green arrows) or decreased (red arrows) in frequency by >2-fold in round 12 after selection at 40 mM Mg2+ relative to round 8 after selection at 80 mM Mg2+. The black arrow at position 642 indicates that the mutations shown at this position were fixed in 99% of the population in round 12. Greek letters with sequence delineated below indicate motifs involved in long-range tertiary structure interactions.
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pgen.1005422.g007: Mutational fitness map of the Ll.LtrB intron during directed evolution in human cells.Secondary structure diagram of the Ll.LtrB intron showing mutation frequencies based on Pacific Biosciences RS circular consensus sequencing after 8 and 12 cycles of directed evolution in HEK-293 cells in culture medium supplemented with 80 mM or 40 mM MgCl2. Round 8 was assessed with 1,395 full-length reads, with the results presented as a color-coded heat map on a secondary structure diagram of the Ll.LtrB intron. Dark to light blue ovals represent conserved nucleotide positions with mutations present in 0–0.3% of the population. Pink to red ovals indicate mutable nucleotide positions with mutations present in >0.3–60% of the population. Green triangles with nucleotide inscribed, indicate positive selection in rounds 1–8 with the indicated nucleotide comprising >80% of the mutations at that position. Round 12 was assessed with 3,069 full-length reads with large colored arrows indicating positions at which the indicated mutation increased (green arrows) or decreased (red arrows) in frequency by >2-fold in round 12 after selection at 40 mM Mg2+ relative to round 8 after selection at 80 mM Mg2+. The black arrow at position 642 indicates that the mutations shown at this position were fixed in 99% of the population in round 12. Greek letters with sequence delineated below indicate motifs involved in long-range tertiary structure interactions.

Mentions: We first sequenced retrohomed introns from round 8 (NCBI SRA database, accession number SAMN03342363) and generated a fitness map that displays the degree of conservation of each nucleotide as a heat map on a secondary structure diagram of the Ll.LtrB intron (Fig 7). The degree of conservation of different nucleotides displayed a wide range and is shown with a scale ranging from dark to light blue for conserved sites (0–0.3% mutations) and from pink to red for mutable sites (>0.3–51% mutations) (Fig 7). On average, the round 8 mutant pool contained 4.4 mutations per intron. The majority of nucleotides (551 of 776) in the intron were conserved (dark or light blue) over eight cycles of directed evolution. Regions required for ribozyme activity (e.g., the catalytic triad in DV, J2/3, which interacts with DV to form the active site, the branch-point A residue in DVI, and the 5’ and 3’ ends of the intron) were invariant, with the exception of a few nucleotides previously shown to be less constrained within those regions (e.g., the dinucleotide bulge in DV). The most variable regions were DIVb, which lies outside the catalytic core, and the two terminal loops of DII. DIVa, which contains a high-affinity LtrA-binding site, showed strong conservation of most nucleotides found to be critical for LtrA binding (positions 557, 559, 561–564), but not position 556 [58,59]. A mutation at position 548 in an internal loop in DIVa was positively selected (green triangle) and could affect LtrA binding.


Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.

Truong DM, Hewitt FC, Hanson JH, Cui X, Lambowitz AM - PLoS Genet. (2015)

Mutational fitness map of the Ll.LtrB intron during directed evolution in human cells.Secondary structure diagram of the Ll.LtrB intron showing mutation frequencies based on Pacific Biosciences RS circular consensus sequencing after 8 and 12 cycles of directed evolution in HEK-293 cells in culture medium supplemented with 80 mM or 40 mM MgCl2. Round 8 was assessed with 1,395 full-length reads, with the results presented as a color-coded heat map on a secondary structure diagram of the Ll.LtrB intron. Dark to light blue ovals represent conserved nucleotide positions with mutations present in 0–0.3% of the population. Pink to red ovals indicate mutable nucleotide positions with mutations present in >0.3–60% of the population. Green triangles with nucleotide inscribed, indicate positive selection in rounds 1–8 with the indicated nucleotide comprising >80% of the mutations at that position. Round 12 was assessed with 3,069 full-length reads with large colored arrows indicating positions at which the indicated mutation increased (green arrows) or decreased (red arrows) in frequency by >2-fold in round 12 after selection at 40 mM Mg2+ relative to round 8 after selection at 80 mM Mg2+. The black arrow at position 642 indicates that the mutations shown at this position were fixed in 99% of the population in round 12. Greek letters with sequence delineated below indicate motifs involved in long-range tertiary structure interactions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524724&req=5

pgen.1005422.g007: Mutational fitness map of the Ll.LtrB intron during directed evolution in human cells.Secondary structure diagram of the Ll.LtrB intron showing mutation frequencies based on Pacific Biosciences RS circular consensus sequencing after 8 and 12 cycles of directed evolution in HEK-293 cells in culture medium supplemented with 80 mM or 40 mM MgCl2. Round 8 was assessed with 1,395 full-length reads, with the results presented as a color-coded heat map on a secondary structure diagram of the Ll.LtrB intron. Dark to light blue ovals represent conserved nucleotide positions with mutations present in 0–0.3% of the population. Pink to red ovals indicate mutable nucleotide positions with mutations present in >0.3–60% of the population. Green triangles with nucleotide inscribed, indicate positive selection in rounds 1–8 with the indicated nucleotide comprising >80% of the mutations at that position. Round 12 was assessed with 3,069 full-length reads with large colored arrows indicating positions at which the indicated mutation increased (green arrows) or decreased (red arrows) in frequency by >2-fold in round 12 after selection at 40 mM Mg2+ relative to round 8 after selection at 80 mM Mg2+. The black arrow at position 642 indicates that the mutations shown at this position were fixed in 99% of the population in round 12. Greek letters with sequence delineated below indicate motifs involved in long-range tertiary structure interactions.
Mentions: We first sequenced retrohomed introns from round 8 (NCBI SRA database, accession number SAMN03342363) and generated a fitness map that displays the degree of conservation of each nucleotide as a heat map on a secondary structure diagram of the Ll.LtrB intron (Fig 7). The degree of conservation of different nucleotides displayed a wide range and is shown with a scale ranging from dark to light blue for conserved sites (0–0.3% mutations) and from pink to red for mutable sites (>0.3–51% mutations) (Fig 7). On average, the round 8 mutant pool contained 4.4 mutations per intron. The majority of nucleotides (551 of 776) in the intron were conserved (dark or light blue) over eight cycles of directed evolution. Regions required for ribozyme activity (e.g., the catalytic triad in DV, J2/3, which interacts with DV to form the active site, the branch-point A residue in DVI, and the 5’ and 3’ ends of the intron) were invariant, with the exception of a few nucleotides previously shown to be less constrained within those regions (e.g., the dinucleotide bulge in DV). The most variable regions were DIVb, which lies outside the catalytic core, and the two terminal loops of DII. DIVa, which contains a high-affinity LtrA-binding site, showed strong conservation of most nucleotides found to be critical for LtrA binding (positions 557, 559, 561–564), but not position 556 [58,59]. A mutation at position 548 in an internal loop in DIVa was positively selected (green triangle) and could affect LtrA binding.

Bottom Line: Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells.By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+.Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

No MeSH data available.


Related in: MedlinePlus