15 research outputs found
BioID assay set-up.
<p><b>(A)</b> Expression of the promiscuous biotin ligase BirA(R118G) (HB*, orange circle) fused to a protein of interest (designated “bait”, blue circle) results in distant-dependent biotinylation (represented as orange stars) of proteins located in the vicinity of the HB*-bait protein if biotin is supplement to the cell culture. Biotinylated proteins are then affinity captured by magnetic streptavidin beads. Since the HB* fusion constructs contain a hemagglutinin (HA) tag (white circle), the BioID assay can be combined with anti-HA immunoprecipitation followed by streptavidin affintiy purification (SA) to specifically capture biotinylated proteins in the immunoprecipitate. <b>(B, C)</b> Biotinylation by HB* occurs mainly <i>in vivo</i> but not in the lysate, because neither supplementing biotin in extracts containing HB*-UPF1 (B) nor addition of an extract primed with biotin (C) boosts total protein biotinylation. 293T cells expressing either no (1, 2), wild-type BirA (HB)-tagged (3, 4) or HB*-tagged UPF1 (5, 6) were incubated without (1, 3, 5) or with 50 μM biotin (2, 4, 6) for 16 hours before extracts were prepared by gentle hypotonic lysis. In contrast to HB*, HB only biotinylates the specific biotin acceptor peptide and is used here as a negative control. (B) Where indicated, extracts were then supplemented with 50 μM biotin and incubated for 16 hours at 4°C before SA purification. (C) Extracts 1 and 2 were mixed with the HB-UPF1 (3, 4) and HB*-UPF1 (5, 6) expressing extracts followed by incubation for 16 hours at 4°C. The total biotinylation was monitored by probing with Streptavidin 800CW after slot blotting of samples onto nitrocellulose membranes.</p
Lists of proteins identified by BioID using HB*-tagged UPF1, UPF2 and SMG5 as a bait.
<p>The normalized spectral counts obtained with the indicated HB*-tagged bait or the HB* alone (specificity control) are shown for each of the identified proteins. Proteins also detected in the HB* control are marked by a black dot, proteins identified by more than one NMD factor are highlighted by the indicated color code.</p
Interaction of the NMD factors UPF1 and SMG5 with mRNP components and decapping factors.
<p>Western blots were performed to detect proteins that co-immunoprecipitate with H16-UPF1<sup>R</sup> <b>(A)</b> or H16-SMG5<sup>R</sup> <b>(B)</b> overexpressed in 293T cells. To boost the phsphorylation state of proteins, cells were treated with okadaic acid for 3 hours before harvesting and phosphatase (PPase) inhibitors were added to the lysates (lanes 4, 10, 11, 17, 18; highlighted by green retangles). To dephosphorylate proteins, cell lysates were incubated with PPase λ prior to IP (lanes 3, 8, 9, 15, 16; red rectangles). Finally, the RNA dependence of the associated proteins was assessed by RNase A treatment of lysates (lanes 7, 9, 11, 14, 16, 18). Input, unbound (5x10<sup>5</sup> cell equivalents each), and immunoprecipitated material (equivalent to 1x10<sup>7</sup> cells) were separated on SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies against the indicated proteins. Exact postions of overexpressed and endogenous proteins are indicated by white and black triangles, respectively, L denotes signal originating from the antibody light chain and HB bleed-through signal from the HA-BirA protein. For better visibility, a higher intensity scan of the membrane piece is shown in (B) for the XRN1 IP samples.</p
Among the interactors identified by the combined BioID / anti-HA immunoprecipitation approach using HB*-UPF1<sup>R</sup>, HB*-UPF2<sup>R</sup> and HB*-SMG5<sup>R</sup>, mRNP components are enriched.
<p>HB*, HB*-UPF1<sup>R</sup>, HB*-UPF2<sup>R</sup>, and HB*-SMG5<sup>R</sup> were transiently expressed in 293T cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.g003" target="_blank">Fig 3</a>. Protein biotinylation was boosted by addition of 50 μM biotin for six hours before harvesting. From the cell extracts, bait-containing complexes were immunoprecipitated using magnetic anti-HA beads followed by SA capture of the biotinylated proteins in these precipitates. The isolated proteins were resolved by NuPAGE and identified by mass spectrometry. <b>(A)</b> Overrepresented biological processes among the 78 common interactors of HB*-UPF1<sup>R</sup>, HB*-UPF2<sup>R</sup> and HB*-SMG5<sup>R</sup> were identifed by GO analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.g003" target="_blank">Fig 3B</a>. <b>(B)</b> The common interactors identified in the tandem purified samples were analyzed by the PPI spider tool as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.g003" target="_blank">Fig 3C</a>. They enrich significantly in a binary protein-protein interaction network (central grey zone) including the UPF1-UPF2-UPF3B (UPF) complex and the exon-exon junction complex (EJC). An extended network (outside the grey zone) connecting general mRNP components and ribosomal proteins is still significantly enriched (p < 0.01) if a single gap (nods depicted by grey gene names) is tolerated between the identified proteins (nods with pie charts). <b>(C)</b> The proteins identified in the combined BioID/co-IP are enriched in domains and motifs that are typical for RNA-binding proteins. These enriched domains of the InterPro database were identified by GO analysis using the tools on enrichnet.org [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.ref078" target="_blank">78</a>] as described in Materials & Methods.</p
Lists of proteins identified with the combined BioID / anti-HA immunoprecipitation approach using HB*-tagged UPF1, UPF2 and SMG5 as a bait.
<p>The normalized spectral counts obtained with the indicated HB*-tagged bait or the HB* alone (specificity control) are shown for each of the identified proteins. Proteins identified by more than one NMD factor are highlighted by the indicated color code.</p
UPF2 transiently interacts with translation factors.
<p><b>(A)</b> Western blots of anti-HA co-immunoprecipitation experiment from 293T cells overexpressing H16-UPF2<sup>R</sup>. Protein hyperphosphorylation, dephosphorylation and RNase A treatment conditions were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.g007" target="_blank">Fig 7</a>. <b>(B)</b> Western blot of co-immunoprecipitation of endogenous EIF4A2 from 293T cells with an antibody against the n-terminus of EIF4A2 (lanes 3,4, 6,7) either with (lanes 4 and 7) or without RNase A treatment (lanes 3 and 6). Whole IgG was used as a control (lanes 2 and 5). Input, unbound (5x10<sup>5</sup> cell equivalents each) and immunoprecipitated material (equivalent to 1x10<sup>7</sup> cells) was used in (A) and (B). White and black triangles denote the position of the overexpressed and endogenous proteins, respectively. <b>(C-E)</b> Proximity ligation assays (PLA) probing for the effect translation inhibition on the pairwise co-localization of UPF2 and EIF4A2 (C), UPF2 and ribosomal protein RPS2 (D), and UPF2 and SMG1 (E). HeLa Tet-Off TCRβ ter68 cells (clone 2.2) were grown and where indicated treated with puromycin (Puro), cycloheximide (CHX) or arsentite. The antibody pairs for detection are depicted above the images. Omission of one or both primary antibodies controlled for the specificity of the PLA signal.</p
The HB*-tagged UPF1, UPF2, and SMG5 constructs are functionally active in the NMD pathway.
<p><b>(A)</b> Schematic of the TCRβ ter68 NMD reporter construct stably integrated in HeLa Tet-Off cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.ref024" target="_blank">24</a>]. The TRE-tight promoter (thick black arrow), the transcription start site (thin arrow) and the SV40 polyadenylation site (black box with flag) are indicated. AUG denotes the beginning of the open reading frame (box), and its sites of premature and normal termination are marked by the premature termination codon (PTC) and the termination codon (TC), respectively. The two introns are depicted as kinked lines <b>(B)</b> Schematic representation of the tested protein constructs. The HA-tag followed by a Gly<sub>16</sub> spacer is shown as white box, BirA as orange box (with the R118G mutation denoted as white star) and bait factors as blue boxes <b>(C-E)</b> Knockdown of the indicated NMD factors was achieved by transiently transfecting the HeLa Tet-Off TCRβ ter68 cells with shRNA expressing plasmids and rescue of NMD activity was attempted by co-transfection of the expression plasmids encoding the indicated RNAi resistant (denoted <sup>R</sup>) constructs. Relative TCRβ ter68 RNA levels, normalized to ACTB mRNA, were measured by real-time qRT-PCR (left side) and quantified by the ΔΔCT method. The columns represent the average expression level of three independent biological replicates. Error bars mark the cumulated variation among the ΔΔCT values in the biological replicates. The levels of the indicated proteins were analyzed by western blotting (right side). (C) Knockdown of endogenous UPF1 can be rescued by expressing H16-UPF1<sup>R</sup>, HB-UPF1<sup>R</sup> or HB*-UPF1<sup>R</sup> fusion proteins, (D) knockdown of endogenous UPF2 can be rescued by expressing H16-UPF2<sup>R</sup>, HB-UPF2<sup>R</sup> or HB*-UPF2<sup>R</sup> fusion proteins, and (E) knockdown of endogenous SMG5 can be rescued by expressing H16-SMG5<sup>R</sup>, HB-SMG5<sup>R</sup>, and HB*-SMG5<sup>R</sup> fusion proteins. Positions of recombinant and endogenous proteins on the blots are indicated on the left by white and black triangles, respectively.</p
BioID using HB*-UPF1<sup>R</sup>, HB*-UPF2<sup>R</sup> and HB*-SMG5<sup>R</sup> captures known and novel interactors.
<p><b>(A)</b> HB*, HB*-UPF1<sup>R</sup>, HB*-UPF2<sup>R</sup>, and HB*-SMG5<sup>R</sup> were transiently expressed in 293T cells at similar levels as the endogenous factor. On the western blots, the HB*-UPF1<sup>R</sup>, HB*-UPF2<sup>R</sup> and HB*-SMG5<sup>R</sup> fusion proteins can be distinguished from their respective endogenous proteins by their slower migration. The biotinylated proteins were isolated by streptavidin affinity capture and analyzed by western blotting for interaction among the NMD factors indicated on the left. The same samples were also analyzed by mass-spectrometry. Putative interactors were identified by excluding proteins that were not detected by more than two times the total normalized spectra counts in isolates of at least one HB*-bait factor when compared to the control isolates from cells expressing free HB*. <b>(B)</b> Overrepresented biological processes in the combined dataset were identified by Gene ontology (GO) analysis performed with enrichnet.org [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.ref078" target="_blank">78</a>]. Only significantly enriched parental GO terms are shown (Fisher q-value ≤ 0.05). Grey bars represent GO term enrichment (frequency in query / frequency in human genome), black points represent the corrected Fisher q-value. <b>(C)</b> The identified putative interactors were analyzed by the PPI spider tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150239#pone.0150239.ref034" target="_blank">34</a>] for the enrichment of a protein-protein subnetwork using the experimental data in the IntAct database. This analysis revealed the significantly enriched (p < 0.01) network of binary interactors. The factors are depicted as pie chart. The diameter of the pie charts is proportional to the logarithmic total normalized spectrum count obtained from the LC-MS/MS analysis. The segments correspond to the fraction of the total normalized spectrum counts from the individual BioID samples for this factor. The connecting lines between the factors are colour-coded according to samples in which they were detected. Black lines denote direct interactions and grey lines connect human factors whose interaction is documented in the IntAct database.</p
Genotoxic stress modifies alternative splicing of endogenous genes.
<p>A. SH-SY5Y cells were incubated with vehicle or with PQ as described in Material and Methods. The bar graph represents the quantification of the RT-PCR splicing analysis of the alternatively spliced regions of the APAF1 (exon 18, e18), H-RAS (exon 5, e 5), ERCC1 (exon 8, e 8), SKP2 (exon 11, e 11), and BIN1 (exon 14, e14) transcripts. The indicated splice forms were subcloned and sequenced to verify their identity. The inclusion or the skipping of variable exons after PQ treatment (black bars) was normalized relative to that observed in the respective controls (light grey bars). Error bars indicate the standard error three biological replicates. The asterisks represents the result of two-tailed t-test: *** indicates p<0.001, ** indicates p<0.01. B. RT-PCR splicing analysis of the alternatively spliced exon 18 (e18) of the APAF1 transcript in SH-SY5Y cells treated with vehicle (control), PQ, or γ-radiation. The asterisks represents the result of one-way ANOVA and Tukey's post test: * indicates p<0.05. C. RT-PCR splicing analysis of the alternatively spliced exon 5 (e5) of the H-RAS transcript in SH-SY5Y cells treated with vehicle, PQ, or cisplatin as described in Material and Methods. The asterisks represents the result of one-way ANOVA and Dunnett's post test: *** indicates p<0.001, ** indicates p<0.01.</p
Genotoxic stress induces nuclear accumulation and phosphorylation of SRPK2.
<p>A. PQ induces DNA damage. Representative confocal micrographs of SH-SY5Y cells treated with PQ and fixed at the indicated time-points. Cells were stained with an anti-γH2AX antibody (<i>upper row</i>) or with an antibody specific for SRPK2 (<i>lower row</i>). B. Inhibition of PQ-induced H2AX phosphorylation by roscovitine. Representative confocal micrographs of SH-SY5Y cells incubated with 10 µM roscovitine and PQ treatment. ãH2AX was detected by immunocytochemistry; nuclei were stained with DAPI. C. Inhibition of the DDR blocks nuclear localization of SRPK2. Representative confocal micrographs of control SH-SY5Y cells (<i>first row</i>), or SH-SY5Y cells incubated with PQ alone (<i>second row</i>), or with PQ and with 10 µM roscovitine (<i>third row</i>), or with PQ and 10 mM caffeine (<i>forth row</i>). SRPK2 was detected by immunocytochemistry; nuclei were stained with DAPI. D. Genotoxic treatments induce nuclear localization of SRPK2. Representative confocal micrographs of untreated SH-SY5Y cells (<i>upper row</i>) or cells treated with 20 µM cisplatin for 18 h (<i>middle row</i>) or irradiated with 10 Gy (<i>lower row</i>) were stained with DAPI and with an anti-SRPK2 antibody. E. Genotoxic treatments induce hyperphosphorylation of SRPK2. Western blots of total cell extract prepared from untreated SH-SY5Y cells or from cells treated with PQ, or with cisplatin for the indicated times. The blot was probed with an anti-SRPK2 antibody. Actin was used as loading control. The slower migrating band is indicated by an arrow.</p