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Articles

RNA Editing in Trypanosoma brucei Requires Three Different Editosomes

Jason Carnes, James Raffaello Trotter, Adam Peltan, Michele Fleck, Kenneth Stuart
Jason Carnes
1Seattle Biomedical Research Institute, Seattle, Washington 98109
2Department of Pathobiology, University of Washington, Seattle, Washington 98195
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James Raffaello Trotter
1Seattle Biomedical Research Institute, Seattle, Washington 98109
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Adam Peltan
3Immunology and Infection Unit, Department of Biology, University of York, Heslington, York YO10 5YW, United Kingdom
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Michele Fleck
1Seattle Biomedical Research Institute, Seattle, Washington 98109
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Kenneth Stuart
1Seattle Biomedical Research Institute, Seattle, Washington 98109
2Department of Pathobiology, University of Washington, Seattle, Washington 98195
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  • For correspondence: kstuart@u.washington.edu
DOI: 10.1128/MCB.01374-07
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  • FIG. 1.
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    FIG. 1.

    KREN3 repression inhibits growth. Growth of the BF RKO-KREN3 cells in the presence of tet (•), which allows KREN3 expression, or without tet (▦), which represses KREN3 expression. Growth eventually resumes due to the loss of tet regulation and reexpression of KREN3.

  • FIG. 2.
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    FIG. 2.

    KREN3 repression does not alter editosome sedimentation. Western analysis of lysates of BF RKO-KREN3 cells that were grown with (KREN3 expressed) or without (KREN3 repressed) tet for 3 days. Fractions from glycerol gradients were probed with a mixture of monoclonal antibodies that are specific for KREPA1, KREPA2, KREL1, and KREPA3 editosome proteins. Fractions 6 to 8 are the ∼20S peak of the gradient. A ∼20S fraction from purified PF mitochondria, mito(+), is used as a positive control.

  • FIG. 3.
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    FIG. 3.

    KREN3 repression does not alter in vitro editing activities on an A6-derived substrate. The ∼20S glycerol gradient fractions from BF RKO-KREN3 cells grown for 3 days in the presence (E, expressed) or absence (R, repressed) of tet were incubated with A6-derived RNA substrates to assay endonucleolytic cleavage of insertion (A) or deletion (B) and precleaved insertion (C) or deletion (D) editing activities. The RNA substrates and products are shown schematically with the asterisk indicating radiolabel and the wedge indicating the cleavage site. The cleavage products in panels A and B (arrows) are due to RNA editing endonuclease activity, as indicated by the requirement for gRNA (-guide). Lanes with radiolabeled substrate (input) or positive control ∼20S glycerol gradient fractions from PF lysates [20S(+)] are indicated. The amounts of fractions used in panels C and D are indicated in microliters above each lane. T1-digested substrate RNA is used as a marker to localize the cleavage site.

  • FIG. 4.
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    FIG. 4.

    KREN3 repression inhibits COII editing in vivo. Real-time PCR analysis comparing total RNA from BF RKO-KREN3 cells in which KREN3 expression was repressed for 3 days with RNA from cells in which KREN3 was expressed. The relative change of each target amplicon is determined in triplicate with either β-tubulin (left bar) or 18S rRNA (right bar) as an internal control. Preedited mRNA is indicated by white bars, edited mRNA is indicated by dark gray bars, mRNA for RNase III-motif-containing proteins is indicated by black bars, and mRNA that is never edited is indicated by light gray bars. On this log-scale graph, 1 indicates no change in relative amount of RNA, while bars above 1 indicate an increase and bars below 1 indicate a decrease in relative RNA amount.

  • FIG. 5.
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    FIG. 5.

    KREN3 repression eliminates COII endonuclease activity in vitro. COII-derived cis (A) and trans (B) substrate RNAs with the radiolabel and cleavage site indicated by an asterisk and wedge, respectively. Cleavage assays with cis substrate RNA COIIcisU1 using ∼20S glycerol gradient fractions of lysates from BF RKO-KREN3 (N3), BF RKO-KREN1 (N1), or BF RKO-KREN2 (N2) cells grown for 3 days in the presence (E, expressed) or absence (R, repressed) of tet (C) or purified PF TAP-tagged editosomes (D). The cleavage product (arrow) is mapped relative to alkaline hydrolysis (OH) and T1-digested substrate RNA (T1) ladders. “mito (+)” is an ∼20S fraction from purified PF mitochondria used as a positive control, and “water (−)” is used as a negative control. Note that cleavage only occurs in editosomes that contain KREN3. Western analysis of the TAP-tagged editosomes with monoclonal antibodies against KREPA1, KREPA2, KREL1, and KREPA3 editosome proteins (F) and with antibody against the CBP tag upon reprobing the stripped blot (G). Cleavage assays with the trans substrate RNA using the TAP-tagged editosomes (E). Loss of cleavage upon omission of gRNA (-guide) shows that cleavage is due to RNA editing endonuclease activity.

  • FIG. 6.
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    FIG. 6.

    Key residues in the RNase III motif of KREN3 are required for growth and COII-derived substrate RNA cleavage in vitro. (A) Growth of RKO-WTN3-β, RKO-E197V-β, RKO-D204A-β, and RKO-E271A-β cell lines with the Reg-KREN3 allele expressed (E) or repressed (R), as indicated in the inset. The D204A and E271A alleles fail to rescue for loss of KREN3, while the E197V and WT allele do rescue. (B) Western analysis of whole-cell lysates from BF RKO-KREN3 (RKO), RKO-WTN3-β, RKO-E197V-β, RKO-D204A-β, and RKO-E271A-β cells probed with monoclonal antibodies against KREPA1, KREPA2, KREL1, and KREPA3. This shows that an equivalent amount of material was used in each affinity purification. (C) Western of TEV eluates after IgG affinity purification from lysates shown in panel B using monoclonal antibodies against KREPA1, KREPA2, KREL1, and KREPA3. (D) The blot from panel C stripped and reprobed with antibody against CBP tag on KREN3 proteins. (E) COII-derived substrate RNA cleavage by TEV eluates examined in panels C and D. The cleavage product (arrow) is localized relative to alkaline hydrolysis (OH) and T1-digested substrate RNA (T1). Note that editosomes with WT KREN3 cleave COII-derived substrate RNA, while those with mutant KREN3 do not. “Mito (+)” is a positive control lane of ∼20S fraction from purified PF mitochondria, while omission of editosomes [water (−)] or tagged editosomes (RKO) are negative controls.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 - Fig. S1 (Sequence alignment of RNase III signature motifs with KREN3)
      Zipped JPEG document, 164K.
    • Supplemental file 2 - Legend to Fig. S1
      MS Word document, 24K.
    • Supplemental file 3 - Supplemental text
      MS Word document, 45K.
    • Supplemental file 4 - Table S1 (Oligonucleotide sequences used for cloning and real-time PCR)
      MS Word document, 51K.
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RNA Editing in Trypanosoma brucei Requires Three Different Editosomes
Jason Carnes, James Raffaello Trotter, Adam Peltan, Michele Fleck, Kenneth Stuart
Molecular and Cellular Biology Dec 2007, 28 (1) 122-130; DOI: 10.1128/MCB.01374-07

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RNA Editing in Trypanosoma brucei Requires Three Different Editosomes
Jason Carnes, James Raffaello Trotter, Adam Peltan, Michele Fleck, Kenneth Stuart
Molecular and Cellular Biology Dec 2007, 28 (1) 122-130; DOI: 10.1128/MCB.01374-07
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KEYWORDS

Organelles
RNA editing
Trypanosoma brucei brucei

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