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