The highly related DEAD box RNA helicases p68 and p72 exist as heterodimers in cells

Abstract

The RNA helicases p68 and p72 are highly related members of the DEAD box family of proteins, sharing 90% identity across the conserved core, and have been shown to be involved in both transcription and mRNA processing. We previously showed that these proteins co-localise in the nucleus of interphase cells. In this study we show that p68 and p72 can interact with each other and self-associate in the yeast two-hybrid system. Co-immunoprecipitation experiments confirmed that p68 and p72 can interact in the cell and indicated that these proteins preferentially exist as hetero-dimers. In addition, we show that p68 can interact with NFAR-2, a protein that is also thought to function in mRNA processing. Moreover, gel filtration analysis suggests that p68 and p72 can exist in a variety of complexes in the cell (ranging from approximately 150 to approximately 400 kDa in size), with a subset of p68 molecules being in very large complexes (>2 MDa). The potential to exist in different complexes that may contain p68 and/or p72, together with a range of other factors, would provide the potential for these proteins to interact with different RNA substrates and would be consistent with recent reports implying a wide range of functions for p68/p72.

Figure 1

Interaction of p68, p72 and fibrillain in the yeast two-hybrid system. cDNAs encoding p68, p72 or fibrillarin were cloned in the yeast two-hybrid vectors (Clontech) such that they were fused either to the GAL4 DNA binding domain (pAS2) or to the transcriptional activation domain (pACT2) and checked for interaction in a standard yeast two-hybrid assay. +++, strong interaction, 100% blue colonies in <30 min; ++, intermediate interaction, >50% blue colonies in <4 h; +/–, weak interaction, <50% blue colonies in 24 h; –, no interaction, no blue colonies. Fib., fibrillarin; NEAD, p68 and p72 in which the first aspartate (D) in the D-E-A-D motif had been mutated to asparagine (N) giving ATPase and helicase inactive proteins.

Figure 2

Deletion mapping of the regions in p68 interacting with p68 and p72. In each case deletion derivatives were tested for interaction with the respective full-length partners in the yeast two-hybrid system. The amino acids included in each of the deletions are indicated as are the conserved ‘DEAD box’ motifs. FL, full length. +++, strong interaction, 100% blue colonies in <30 min; ++, intermediate interaction, >50% blue colonies in <4 h; +/–, weak interaction, <50% blue colonies in 24 h; –, no interaction, no blue colonies.

Figure 3

Deletion mapping of the regions in p72 interacting with p68 and p72. In each case deletion derivatives were tested for interaction with the respective full-length partners in the yeast two-hybrid system. The amino acids included in each of the deletions are indicated as are the conserved ‘DEAD box’ motifs. FL, full length. +++, strong interaction, 100% blue colonies in <30 min; ++, intermediate interaction, >50% blue colonies in <4 h; +/–, weak interaction, <50% blue colonies in 24 h.

Figure 4

Analysis of p72 species in cell lines and interaction with p68. (A) Western blotting of 293 cell lysates with an antibody generated against a p72 peptide detects three protein species. (B) Immunoprecipitation/ western blotting experiments to examine p68/p72 interactions. Proteins from 293 cell lysates were immunoprecipitated with p72 antibodies 43 and 44 (lanes 1 and 2), p68 antibody 2906 (lane 3), or an irrelevant antibody (lane 4) and then western blotted with the p72 antibody 43. p72, p82 and potential p72/p82 (p72/p82?) species and heavy chain cross-reaction (H) are indicated. The antibodies used for immunoprecipitation and western blotting were rabbit polyclonals, hence the strong cross-reaction for the heavy chain.

Figure 5

Analysis of interaction between NFAR-1/NFAR-2 and p68/p72 in the yeast two-hybrid system. (A) Diagrammatic representation of NFAR-1 and NFAR-2 coding regions highlighting identical regions (amino acids 1–687) and differences resulting from alternative splicing. (B) Interactions between full-length and deletion derivatives of NFAR-1/NFAR-2 and p68/p72. +++, strong interaction, 100% blue colonies in <30 min; ++, intermediate interaction, >50% blue colonies in <4 h; +/–, weak interaction, <50% blue colonies in 24 h; –, no interaction, no blue colonies. (C) The regions of p68 and p72 which show the strongest interaction with NFAR-2.

Figure 6

Co-immunoprecipitation of p68 and p72 from cell lysates. (A) Western blot of untransfected and myc-p72 transfected 293 cell lysates with a p68-specific antibody (2906) showing co-immunoprecipitation of exogenously expressed myc-tagged p72 with endogenous p68 (lanes 1, 2 and 4, lysates from cells transfected with myc-p72; lane 3, untransfected cells). Lane 1, endogenous p68 in cell lysate; lane 2, immunoprecipitation of myc-p72 with the anti-myc antibody 9E10; lane 3, immunoprecipitation of proteins from untransfected cells with 9E10; lane 4, immunoprecipitation of endogenous p68 from myc-p72 transfected cells with the p68-specific antibody PAb 204. H, cross-reaction of heavy chain. (B) Western blot of cells transfected with GST-tagged p68/p72 (and GST vector control) with a p68-specific antibody (2906) showing co-immunoprecipitation of exogenously expressed GST-tagged p72 with endogenous p68. In each case GST-tagged proteins were immunoprecipitated with a GST-specific antibody (lanes 1 and 2, cells transfected with GST–tagged vector control; lanes 3 and 4, cells transfected with GST-tagged p68; lanes 5 and 6, cells transfected with GST-tagged p72). Lane 1, lysate showing endogenous p68; lane 2, GST immunoprecipitation; lane 3, lysate showing endogenous and GST-tagged p68; lane 4, GST immunoprecipitation, showing immunoprecipitated GST-tagged p68 and a very low level of endogenous p68; lane 5, lysate showing endogenous p68; lane 6, GST immunoprecipitation showing endogenous p68 immunoprecipitating with GST-tagged p72.

Figure 7

Western blots showing gel filtration elution profiles of p68, p72, p82 and NFAR. p68, p72 and NFAR in the gel filtration fractions were detected by western blotting using appropriate antibodies: 2906 for p68, 43 for p72, 9E10 for myc-tagged p72/p82 and the anti-NFAR antibody for NFAR-1 and NFAR-2. The void volume and elution position of the Pharmacia FPLC size markers are indicated, as are molecular weight markers (in kDa) for all western blots. *Lysates which had been treated with RNase A prior to gel filtration. Note that myc-tagged p72 and p82 have an electrophoretic mobility slightly slower than that of the respective endogenous proteins.

Figure 8

Localisation of p68, p72, fibrillarin and NFAR in HeLa cells as determined by immunofluorescence microscopy. All cells were labelled with DAPI to detect DNA (d, h, i, p, t, x). The relative localisation of proteins was determined by labelling using appropriate secondary antibodies conjugated to FITC (green) and Texas Red, respectively, as follows: (a and b) p68/p72; (e and f) p68/NFAR; (i and j) p72/NFAR; (m and n) fibrillarin/p68; (q and r) fibrillarin/p72; (u and v) fibrillarin/NFAR. The respective merged images are shown in (c), (g), (k), (o), (s) and (w). Co-localisation is indicated by arrowheads. The images in (i–l) are composites of two images since in this case cells transfected with myc-tagged p72 were required. For each pair of proteins the primary antibodies were rabbit polyclonal and mouse monoclonal antibodies, respectively, thus allowing differential FITC/Texas Red staining by the secondary antibodies.