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Collaborator of alternative reading frame protein (CARF) regulates early processing of pre-ribosomal RNA by retaining XRN2 (5'-3' exoribonuclease) in the nucleoplasm.

Sato S, Ishikawa H, Yoshikawa H, Izumikawa K, Simpson RJ, Takahashi N - Nucleic Acids Res. (2015)

Bottom Line: We show that overexpression of CARF increases the localization of XRN2 in the nucleoplasm and a concomitant suppression of pre-rRNA processing that leads to accumulation of the 5' extended from of 45S/47S pre-rRNA and 5'-01, A0-1 and E-2 fragments of pre-rRNA transcript in the nucleolus.This was also observed upon XRN2 knockdown.Knockdown of CARF increased the amount of XRN2 in the nucleolar fraction as determined by cell fractionation and by immnocytochemical analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, United-graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan.

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CARF associates with XRN2. (A) Using anti-FLAG, FLAG–CARF-associated complexes were immunoprecipitated from the extract of TOCARF cells after induction with 1 ng/ml doxycycline (dox). The FLAG–CARF-associated complexes (1 μg protein/lane) were separated on a 7.5% SDS-polyacrylamide gel and visualized with silver staining. Molecular weights (MW) are given on the left. Whole-cell lysate (WCL) were analyzed with immunoblotting with the indicated antibodies (20 μg protein/lane). (B) FLAG–CARF-associated complexes were immunoprecipitated with anti-FLAG from TOCARF cells after induction with doxycycline (lanes 2 and 6, 0 ng/ml; lanes 3 and 7, 0.1 ng/ml; lanes 4 and 8, 10 ng/ml), and the immunoprecipitates from Flp-In T-Rex 293 cells (lanes 1 and 5) were analyzed with immunoblotting with the antibodies indicated on the left (IB). The input fraction for each cell preparation (2 × 105 cells/lane) was also analyzed. (C) HA–XRN2-associated complexes were immunoprecipitated from cells with anti-HA following treatment: induction of CARF expression with 100 ng/ml dox (lanes 1, 3, 4 and 6) and transfection with an HA–XRN2 expression plasmid (lanes 2, 3, 5 and 6). Input fractions (2 × 105 cells/lane) and each complex were analyzed with immunoblotting using the antibodies indicated on the left. (D) Using anti-FLAG, FLAG–CARF-associated complexes were immunoprecipitated from cells that stably expressed FLAG–CARF. Immunoprecipitates were treated without (−) or with (+) RNase A (lanes 6 and 7). Controls were performed with Flp-In T-Rex 293 cells that were untransfected (lanes 4 and 5) and that stably expressed FLAG–fibrillarin (lanes 8 and 9). Input fractions (10 μg protein/lane) and each complex were analyzed with immunoblotting using the antibodies indicated on the left. (E) CARF- and XRN2-associated complexes were immunoprecipitated with anti-CARF and anti-XRN2 from 293T cell lysate (1 mg/1 ml), respectively (lanes 3 and 4). Normal rabbit IgG was used as a control (lane 2). Input fraction (2%) and complexes were analyzed with immunoblotting using the antibodies indicated on the left.
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Figure 1: CARF associates with XRN2. (A) Using anti-FLAG, FLAG–CARF-associated complexes were immunoprecipitated from the extract of TOCARF cells after induction with 1 ng/ml doxycycline (dox). The FLAG–CARF-associated complexes (1 μg protein/lane) were separated on a 7.5% SDS-polyacrylamide gel and visualized with silver staining. Molecular weights (MW) are given on the left. Whole-cell lysate (WCL) were analyzed with immunoblotting with the indicated antibodies (20 μg protein/lane). (B) FLAG–CARF-associated complexes were immunoprecipitated with anti-FLAG from TOCARF cells after induction with doxycycline (lanes 2 and 6, 0 ng/ml; lanes 3 and 7, 0.1 ng/ml; lanes 4 and 8, 10 ng/ml), and the immunoprecipitates from Flp-In T-Rex 293 cells (lanes 1 and 5) were analyzed with immunoblotting with the antibodies indicated on the left (IB). The input fraction for each cell preparation (2 × 105 cells/lane) was also analyzed. (C) HA–XRN2-associated complexes were immunoprecipitated from cells with anti-HA following treatment: induction of CARF expression with 100 ng/ml dox (lanes 1, 3, 4 and 6) and transfection with an HA–XRN2 expression plasmid (lanes 2, 3, 5 and 6). Input fractions (2 × 105 cells/lane) and each complex were analyzed with immunoblotting using the antibodies indicated on the left. (D) Using anti-FLAG, FLAG–CARF-associated complexes were immunoprecipitated from cells that stably expressed FLAG–CARF. Immunoprecipitates were treated without (−) or with (+) RNase A (lanes 6 and 7). Controls were performed with Flp-In T-Rex 293 cells that were untransfected (lanes 4 and 5) and that stably expressed FLAG–fibrillarin (lanes 8 and 9). Input fractions (10 μg protein/lane) and each complex were analyzed with immunoblotting using the antibodies indicated on the left. (E) CARF- and XRN2-associated complexes were immunoprecipitated with anti-CARF and anti-XRN2 from 293T cell lysate (1 mg/1 ml), respectively (lanes 3 and 4). Normal rabbit IgG was used as a control (lane 2). Input fraction (2%) and complexes were analyzed with immunoblotting using the antibodies indicated on the left.

Mentions: To gain insight into the role of CARF in cellular function, we examined proteins associated with CARF using a combination of an epitope-tagged pull-down methodology and LC-MS/MS (28). We used a site-directed (Flp-In) recombinase-based system to generate isogenic cell lines (27) in which a cytomegalovirus promoter was used to drive the expression of CARF that was integrated at a common locus in the Flp-In T-Rex 293 genome (28). A product of a single-copy transgene of CARF was tagged at the amino terminus with a FLAG affinity purification tag that is used for visualization and purification. Using this approach, we obtained isogenic cell lines expressing FLAG-tagged CARF (FLAG–CARF) with a molecular weight of ∼70 kDa, which corresponds to that estimated from its amino acid sequence. FLAG–CARF was localized mostly in the nucleoplasm (Supplementary Figure S1A) as reported (17). We analyzed FLAG–CARF-associated proteins from TOCARF cell lysate using anti-FLAG mAb coupled beads. Associated proteins were separated by SDS–PAGE, visualized by silver staining and several candidate proteins were identified by LC-MS/MS (28). Prominent amongst these was XRN2 (Mascot protein score 1.168, 35 peptides representing 36.5% sequence coverage, Supplementary Table S1). Immunoblot analysis with anti-XRN2 indicated that XRN2 corresponded to a protein band with a molecular weight of ∼100 kDa and most strongly stained among the proteins associated with FLAG–CARF on the SDS–PAGE gel (Figure 1A, arrow). Although mass-based analysis did not identify ARF, it does not necessarily mean that ARF is not there; in a complex mixture, ARF-related peptide ion signals may be masked/sequested by more abundant peptide ion signal, or ARF may be of very low abundance. We know minimally that the structural integrity of recombinant CARF overexpressed in Flp-In T-Rex 293 cells is correct, given that FLAG–CARF can interact with HA-tagged ARF when co-expressed in Flp-In T-Rex 293 cells (Supplementary Figure S1B) (15).


Collaborator of alternative reading frame protein (CARF) regulates early processing of pre-ribosomal RNA by retaining XRN2 (5'-3' exoribonuclease) in the nucleoplasm.

Sato S, Ishikawa H, Yoshikawa H, Izumikawa K, Simpson RJ, Takahashi N - Nucleic Acids Res. (2015)

CARF associates with XRN2. (A) Using anti-FLAG, FLAG–CARF-associated complexes were immunoprecipitated from the extract of TOCARF cells after induction with 1 ng/ml doxycycline (dox). The FLAG–CARF-associated complexes (1 μg protein/lane) were separated on a 7.5% SDS-polyacrylamide gel and visualized with silver staining. Molecular weights (MW) are given on the left. Whole-cell lysate (WCL) were analyzed with immunoblotting with the indicated antibodies (20 μg protein/lane). (B) FLAG–CARF-associated complexes were immunoprecipitated with anti-FLAG from TOCARF cells after induction with doxycycline (lanes 2 and 6, 0 ng/ml; lanes 3 and 7, 0.1 ng/ml; lanes 4 and 8, 10 ng/ml), and the immunoprecipitates from Flp-In T-Rex 293 cells (lanes 1 and 5) were analyzed with immunoblotting with the antibodies indicated on the left (IB). The input fraction for each cell preparation (2 × 105 cells/lane) was also analyzed. (C) HA–XRN2-associated complexes were immunoprecipitated from cells with anti-HA following treatment: induction of CARF expression with 100 ng/ml dox (lanes 1, 3, 4 and 6) and transfection with an HA–XRN2 expression plasmid (lanes 2, 3, 5 and 6). Input fractions (2 × 105 cells/lane) and each complex were analyzed with immunoblotting using the antibodies indicated on the left. (D) Using anti-FLAG, FLAG–CARF-associated complexes were immunoprecipitated from cells that stably expressed FLAG–CARF. Immunoprecipitates were treated without (−) or with (+) RNase A (lanes 6 and 7). Controls were performed with Flp-In T-Rex 293 cells that were untransfected (lanes 4 and 5) and that stably expressed FLAG–fibrillarin (lanes 8 and 9). Input fractions (10 μg protein/lane) and each complex were analyzed with immunoblotting using the antibodies indicated on the left. (E) CARF- and XRN2-associated complexes were immunoprecipitated with anti-CARF and anti-XRN2 from 293T cell lysate (1 mg/1 ml), respectively (lanes 3 and 4). Normal rabbit IgG was used as a control (lane 2). Input fraction (2%) and complexes were analyzed with immunoblotting using the antibodies indicated on the left.
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Figure 1: CARF associates with XRN2. (A) Using anti-FLAG, FLAG–CARF-associated complexes were immunoprecipitated from the extract of TOCARF cells after induction with 1 ng/ml doxycycline (dox). The FLAG–CARF-associated complexes (1 μg protein/lane) were separated on a 7.5% SDS-polyacrylamide gel and visualized with silver staining. Molecular weights (MW) are given on the left. Whole-cell lysate (WCL) were analyzed with immunoblotting with the indicated antibodies (20 μg protein/lane). (B) FLAG–CARF-associated complexes were immunoprecipitated with anti-FLAG from TOCARF cells after induction with doxycycline (lanes 2 and 6, 0 ng/ml; lanes 3 and 7, 0.1 ng/ml; lanes 4 and 8, 10 ng/ml), and the immunoprecipitates from Flp-In T-Rex 293 cells (lanes 1 and 5) were analyzed with immunoblotting with the antibodies indicated on the left (IB). The input fraction for each cell preparation (2 × 105 cells/lane) was also analyzed. (C) HA–XRN2-associated complexes were immunoprecipitated from cells with anti-HA following treatment: induction of CARF expression with 100 ng/ml dox (lanes 1, 3, 4 and 6) and transfection with an HA–XRN2 expression plasmid (lanes 2, 3, 5 and 6). Input fractions (2 × 105 cells/lane) and each complex were analyzed with immunoblotting using the antibodies indicated on the left. (D) Using anti-FLAG, FLAG–CARF-associated complexes were immunoprecipitated from cells that stably expressed FLAG–CARF. Immunoprecipitates were treated without (−) or with (+) RNase A (lanes 6 and 7). Controls were performed with Flp-In T-Rex 293 cells that were untransfected (lanes 4 and 5) and that stably expressed FLAG–fibrillarin (lanes 8 and 9). Input fractions (10 μg protein/lane) and each complex were analyzed with immunoblotting using the antibodies indicated on the left. (E) CARF- and XRN2-associated complexes were immunoprecipitated with anti-CARF and anti-XRN2 from 293T cell lysate (1 mg/1 ml), respectively (lanes 3 and 4). Normal rabbit IgG was used as a control (lane 2). Input fraction (2%) and complexes were analyzed with immunoblotting using the antibodies indicated on the left.
Mentions: To gain insight into the role of CARF in cellular function, we examined proteins associated with CARF using a combination of an epitope-tagged pull-down methodology and LC-MS/MS (28). We used a site-directed (Flp-In) recombinase-based system to generate isogenic cell lines (27) in which a cytomegalovirus promoter was used to drive the expression of CARF that was integrated at a common locus in the Flp-In T-Rex 293 genome (28). A product of a single-copy transgene of CARF was tagged at the amino terminus with a FLAG affinity purification tag that is used for visualization and purification. Using this approach, we obtained isogenic cell lines expressing FLAG-tagged CARF (FLAG–CARF) with a molecular weight of ∼70 kDa, which corresponds to that estimated from its amino acid sequence. FLAG–CARF was localized mostly in the nucleoplasm (Supplementary Figure S1A) as reported (17). We analyzed FLAG–CARF-associated proteins from TOCARF cell lysate using anti-FLAG mAb coupled beads. Associated proteins were separated by SDS–PAGE, visualized by silver staining and several candidate proteins were identified by LC-MS/MS (28). Prominent amongst these was XRN2 (Mascot protein score 1.168, 35 peptides representing 36.5% sequence coverage, Supplementary Table S1). Immunoblot analysis with anti-XRN2 indicated that XRN2 corresponded to a protein band with a molecular weight of ∼100 kDa and most strongly stained among the proteins associated with FLAG–CARF on the SDS–PAGE gel (Figure 1A, arrow). Although mass-based analysis did not identify ARF, it does not necessarily mean that ARF is not there; in a complex mixture, ARF-related peptide ion signals may be masked/sequested by more abundant peptide ion signal, or ARF may be of very low abundance. We know minimally that the structural integrity of recombinant CARF overexpressed in Flp-In T-Rex 293 cells is correct, given that FLAG–CARF can interact with HA-tagged ARF when co-expressed in Flp-In T-Rex 293 cells (Supplementary Figure S1B) (15).

Bottom Line: We show that overexpression of CARF increases the localization of XRN2 in the nucleoplasm and a concomitant suppression of pre-rRNA processing that leads to accumulation of the 5' extended from of 45S/47S pre-rRNA and 5'-01, A0-1 and E-2 fragments of pre-rRNA transcript in the nucleolus.This was also observed upon XRN2 knockdown.Knockdown of CARF increased the amount of XRN2 in the nucleolar fraction as determined by cell fractionation and by immnocytochemical analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biological Science, United-graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan.

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Related in: MedlinePlus