The prolyl isomerase, FKBP25, interacts with RNA-engaged nucleolin and the pre-60S ribosomal subunit

Abstract

Peptidyl-proline isomerases of the FK506-binding protein (FKBP) family belong to a class of enzymes that catalyze the cis-trans isomerization of prolyl-peptide bonds in proteins. A handful of FKBPs are found in the nucleus, implying that the isomerization of proline in nuclear proteins is enzymatically controlled. FKBP25 is a nuclear protein that has been shown to associate with chromatin modifiers and transcription factors. In this study, we performed the first proteomic characterization of FKBP25 and found that it interacts with numerous ribosomal proteins, ribosomal processing factors, and a small selection of chromatin modifiers. In agreement with previous reports, we found that nucleolin is a major FKBP25-interacting protein and demonstrated that this interaction is dependent on rRNA. FKBP25 interacts with the immature large ribosomal subunit in nuclear extract but does not associate with mature ribosomes, implicating this FKBP's action in ribosome biogenesis. Despite engaging nascent 60S ribosomes, FKBP25 does not affect steady-state levels of rRNAs or its pre-rRNA intermediates. We conclude that FKBP25 is likely recruited to preribosomes to chaperone one of the protein components of the ribosome large subunit.

Keywords: FK506 binding protein; nucleolin; nucleus; ribosome biogenesis.

FIGURE 1.

FKBP25 localizes to the nucleus and nucleolus and associates with proteins in these compartments. (A) Coomassie-stained SDS-PAGE of FKBP25-FLAG immunoprecipitations performed from HEK293 cells. (B) Functional annotation and enrichment analysis of FKBP25-interacting proteins identified in two mass spectrometry experiments. (C) Western blots of FKBP25-FLAG immunoprecipitations from HEK293 cells. (D) Cellular fractionation of HEK293 cells displaying the localization of FKBP25 by Western blot. Tubulin is a cytoplasmic marker, H4 is a nuclear/nucleolar marker, and UBF is a nucleolar marker.

FIGURE 2.

FKBP25 interacts with nucleolin RRMs 1 and 2 in the presence of rRNA. (A) GST pulldown assays of GST, GST-FKBP25 N-terminal domain (NTD), GST-FKBP25 PPI and GST-FKBP25 FL against 6-His nucleolin RRM1-4 + RGG. Pulldowns were performed with and without rRNA. (B) GST pulldown assays using GST-NCL RRM1-2 (aa 290–480) (see Supplemental Fig. S4) against full-length 6His-FKBP25 in the presence and absence of RNase and supplemented with total RNA from HEK293 cells. (C) FLAG Immunoprecipitations from HEK293 cellular extract with and without pretreatment of RNase A. (D) Trizol extracted RNA from FLAG immunoprecipitations from HEK293 nuclear material electrophoresed on a denaturing formaldehyde-agarose gel. (E) Schematic of rDNA transcript. The position of primer sets used for qPCR is indicated below the schematic. (F) qPCR analysis of reverse transcribed RNA extracted from FLAG immunoprecipitations. GAPDH is used as an mRNA control.

FIGURE 3.

FKBP25 interacts with the pre-60S ribosome in the presence of nucleolin. (A) Sucrose density gradient ultracentrifugation of HEK293 nuclear extract. RNA and protein were extracted from each fraction and were resolved by denaturing-formaldehyde gel electrophoresis or SDS-PAGE and Western blot. (B) Sucrose density gradient ultracentrifugation of FLAG-FKBP25 immunoprecipitate from HEK293 cells. RNA and protein were extracted from each fraction and were resolved by denaturing formaldehyde gel electrophoresis or SDS-PAGE and Western blot.

FIGURE 4.

Overexpression or knockdown of FKBP25 does not affect steady-state levels of pre-rRNA intermediates. (A) Northern blot of RNA extracted from untransfected HEK293 cells or HEK293 cells transfected with control siRNA or FKBP25-targeting siRNA. Blots were probed for ITS1 (left) or ITS2 (right). Extracts were Western blotted for FKBP25 to verify knockdown. (B) Northern blot of RNA extracted from HEK293 from control and cells overexpressing FLAG-FKBP25. Blots were probed for ITS1 (left) or ITS2 (right). (C) Northern blot of HEK293 cells treated with vehicle or rapamycin probed for ITS1 (left) or ITS2 (right). (D) Schematic of rRNA processing steps. (*) Nonspecific probe binding.