Article Figures & Data
Figures
- Figure 1. Exogenous/synthetic intronization (ESI)-mutagenesis: a one-step recombineering procedure to introduce point mutations in bacterial artificial chromosome (BAC).
(A) Scheme showing the organisation of a eukaryotic gene with exons (in blue) and their flanking introns. Introns are spliced out from pre-messenger RNAs to produce mature messenger RNAs (mRNAs) containing only exons. The core splicing signals constitute the minimal information required for the splicing of an intron, and include a 5′ and 3′ splicing signal sequences (5′SS and 3′SS, respectively), a branch point, and a poly-pyrimidine tract (P.P.T). The position of these signals relative to the exon/intron border is indicated with the corresponding nucleotides shown in pink (in introns) or light orange (in exons). ESI mutagenesis is a one-step recombineering procedure that relies on the introduction into a BAC of a synthetic intron coding a selectable marker along with the mutation of interest, thereby allowing for the easy selection of correct recombinants. After transcription of the transgene in eukaryotic cells, the synthetic intron is spliced out to produce a mutated mRNA. (B) The synthetic intron constitutes of the intronic core splicing signal (in pink), an antibiotic resistance cassette (Atb.R) under the control of a dual eukaryotic/prokaryotic promoter (in green), and two loxP sites flanking the antibiotic resistance cassette. To serve as a template for BAC recombineering, the synthetic intron is amplified by PCR with primers containing ∼50-bp-long homology arms to the targeted sequence (in blue), the mutation(s) to introduce (in orange), and the exonic part of the 5′SS and 3′SS (in light orange). (C) Scheme showing the introduction of a point mutation into a BAC by ESI mutagenesis. Depending on the position of the sequence to mutate, the point mutation can be inserted along with the synthetic intron into an exon (left panel), or if the mutation is located in the proximity of a pre-existing intron, the part of the synthetic intron encoding the antibiotic resistance can be targeted into this intron (right panel). The synthetic intron is spliced out during RNA maturation, and only the desired mutation is present in the mRNA.
- Figure S1. Available synthetic intron cassettes.
(A, B, C, D) The synthetic intron cassettes generated in this study are depicted (A, B, C, D). The resistance genes and the promoters (bacterial and eukaryotic) controlling their expression are shown. When present, the loxP sites allow the removal of the antibiotic resistances by the CRE recombinase. The position of the core intron-based splicing signal sequences (intronic splicing donor site, branch point, polypirimidine tract (PPT), and intronic splicing acceptor site) is shown. The plasmid from which each cassette can be amplified is indicated. Numbers indicate the nucleotide positions within the cassette.
- Figure S2. Exogenous/synthetic intronization mutagenesis of the GTSE1 SxIP motifs.
(A) Scheme representing the sequence of exon 9 of GTSE1 containing the two SxIP motifs (in orange) and a potential insertion site for the synthetic intron (in pink). Distance (in bp) of the potential insertion site for the synthetic intron to the border with introns 8–9 and 9–10 are presented. (B) Primers used to amplify the synthetic intron are presented. They are composed of (i) homology arms to GTSE1, (ii) the SxIP mutations (in orange), and (iii) a synonymous mutation (in orange) allowing the potential insertion site (in pink) to fit the CAGG consensus. (C) The amplified synthetic intron is recombined onto the GTSE1 bacterial artificial chromosome. The synthetic intron is spliced out from the pre-mRNA to yield a mRNA coding GTSE1 mutated at both SxIP motifs.
- Figure S3. The exogenous/synthetic intronization (ESI) mutagenesis procedure at a glance.
(A) Scheme presenting the bacterial artificial chromosome (BAC) recombineering protocol. Important parameters to pay attention to are indicated. (B) Scheme showing the procedure to obtain the ESI mutagenesis BAC recombineering template. Three PCRs (see Figs 1B and S1 for primer design) are performed to amplify the synthetic intron and pooled for subsequent PCR purification. The resulting product is used as recombineering template. (C) The ESI-mutated BAC is transfected into eukaryotic cells. Stably transfected cells are selected using the antibiotic resistance coded by the synthetic intron. If the BAC carries a fluorescent protein tagged transgene, the antibiotic resistance associated to the tag should be use for selection.
- Figure 2. Exogenous/synthetic intronization (ESI)-mutated bacterial artificial chromosome (BAC) transgenes yield proteins of the right size and localization.
(A) GFP-tagged BAC transgenes (Parental BAC, P) were mutated by ESI mutagenesis to be RNAi resistant (ESI-mutated BAC, ESI) and transfected into HeLa cells. (B, C, D) Depending on the position of the sequence to mutate, the synthetic intron was either targeted into an exon (left panel, light blue label in B, C, and D) or into the neighbouring intron by making use of preexisting 5′SS, and/or 3′SS (right panel, light brown label in B, C, and D). Names of the mutated transgene are indicated in the panel corresponding to their mutation strategy. The ESI-mutated AURKA transgene is analysed in Fig 4. The GTSE1 transgene was ESI mutated at its SxIP motifs and is analysed in Fig 3. (C) GFP-tagged BAC transgenes ESI mutated to be RNAi resistant yield proteins of the expected size. Immunoblotting on cell lysate of pools of HeLa cells transfected with either parental BACs (P) or ESI-mutated BACs (ESI). GFP antibody was used as a probe. Transgene names are indicated at the top. Red asterisks mark the predicted size of the GFP-tagged protein. (D) GFP-tagged transgenes ESI mutated to be RNAi resistant show the same cellular localization as their parental GFP-tagged transgenes. Still images of live cell imaging on HeLa stably transfected with the parental or the ESI-mutated BAC. CHMP4B and AURKB-GFP transgenes were detected by immunofluorescence with anti-GFP antibody. Because of differences in expression level within cell pool, each picture was acquired and scaled independently of the others. Scale Bar 10 μm.
Source data are available for this figure.
- Figure 3. Exogenous/synthetic intronization (ESI)-mutated bacterial artificial chromosome (BAC) transgenes show the expected phenotype.
(A) BAC transgenes ESI mutated to be RNAi resistant show RNAi resistance at the mRNA level. HeLa wild type, parental BAC lines, and ESI-mutated BAC lines were treated with control-siRNA or transgene-specific siRNAs. Levels of mRNA corresponding to the transgenes were determined by qPCR and normalized to GAPDH mRNA levels. For each cell lines, the % of mRNA remaining in the transgene-siRNA treated cells as compared with the control-siRNA treated cells are presented (N = 2 exp.). A panel of three transgenes mutated by targeting the synthetic intron into an exon (light blue label) and three transgenes mutated by targeting the synthetic intron into a preexisting intron (light brown label) were analysed. Error bars represent SD. (B) BAC transgenes ESI mutated to be RNAi resistant show RNAi resistance at the protein level. HeLa and clonal HeLa lines expressing ESI-mutated CEP135 or AURKB-GFP–tagged BAC transgenes were treated with control, CEP135 or AURKB-siRNAs. Levels of AURKB, CEP135, and Tubulin were monitored by immunoblot using specific antibodies. (C) Mutation of GTSE1 SxIP motifs into SxNN by ESI mutagenesis disrupts the interaction of a GTSE1-GFP BAC transgene with EB1. Cell lysates from U2OS, U2OS expressing an ESI-mutated GTSE1-GFP SxNN BAC transgene, and U2OS expressing an endogenously GFP-tagged GTSE1 (GTSE1-GFP Cas9) were used in pull-downs with GST or GST-EB1 as baits. Pull-downs inputs and outputs were probed by immunoblot using GTSE1 antibody. GST and GST-EB1 fusion were visualized by Coomassie Blue.
- Figure 4. An AURKA-GFP bacterial artificial chromosome transgene exogenous/synthetic intronization (ESI)–mutated to carry RNAi resistance rescues endogenous AURKA depletion.
(A) An AURKA-GFP transgene ESI mutated to be RNAi resistant is expressed at endogenous level and is RNAi resistant. Immunoblotting on U2OS and a U2OS clone expressing the ESI-mutated AURKA-GFP bacterial artificial chromosome transgene (U2OS AURKA-GFP siRES [ESI]), both treated with control- or AURKA-siRNA and blocked in mitosis. Antibodies against AURKA and Tubulin were used as probe. (B, C) The ESI-mutated AURKA-GFP transgene shows the correct cellular localization. Immunofluorescence on U2OS and U2OS AURKA-GFP siRES (ESI) treated with control- and/or AURKA-siRNA. (B, C) AURKA antibody (B) or AURKA and Tubulin antibodies (C) were used as a probe. (D, E, F) The ESI-mutated AURKA-GFP transgene rescues the mitotic arrest (D) and spindle collapse (E, F) observed after endogenous AURKA depletion. (D) The percentage of cells in each mitotic phase is presented (D) for U2OS and U2OS AURKA-GFP siRES (ESI) clone treated with control- or AURKA-siRNA (N = 3 exp; error bars represent standard error). (E) Metaphase U2OS and U2OS AURKA-GFP siRES (ESI) cells were treated with control- or AURKA-siRNA and stained by immunofluorescence using DAPI and an antibody against CEP135 (E). (F) The pole-to-pole distance (μm) in metaphase cells is presented (F). In AURKA-depleted U2OS, spindle length was measured in prometaphase cells with nearly aligned chromosomes. The number of mitotic cells used in each condition (n) and statistically significant differences to U2OS treated with control-siRNA are indicated (Kruskal–Wallis, followed by paired Wilcoxon test). (G) Schematic showing the construction of the GTSE1-14A mutant with an artificial intron. (H) Cells containing the GTSE1-14A mutation have a defect in chromosome segregation. Images on the left show normal versus “lagging” chromosomes. Histogram shows the quantification of lagging chromosomes in indicated cell lines/conditions. n ≥ 143 cells from three independent experiments. Error bars indicate SEM. P-values from Mann–Whitney test. All scale bars 5 µm.
- Figure S4. Bacterial artificial chromosome (BAC) transgenes harbouring an artificial intron are correctly spliced.
RT-qPCR absolute quantification of mRNA levels of BAC transgenes expressed in U2OS cells. The number of copies per PCR was calculated according to the corresponding standard curve generated by plotting the quantification cycle (Cq) values against the logarithm of the quantities of standard samples. All values were normalized to GAPDH. Endogenous genes were silenced by RNAi. (A) RT-qPCR absolute quantification of GFP-tagged BAC transgene AURKA expression (AURKA-GFP siIRES [exogenous/synthetic intronization]). Amplified regions are indicated. Control (primer pair GV3247/GV3248): amplifies a PCR product in both, correctly and incorrectly spliced RNA. All spliced correctly (primer pair: GV3249/GV3250): amplifies a PCR product only in the correctly spliced gene. All splice failure: (primer pair: GV3251/GV3250) amplifies a PCR product only in incorrect spliced RNA. (B) RT-qPCR absolute quantification of GFP-tagged BAC transgene GTSE114A expression. Control (primer pair: GV3252/GV3253): amplifies a PCR product in both, correctly and incorrectly spliced RNA. All spliced correctly (primer pair: GV3254/GV3256): amplifies a PCR product only in the correctly spliced RNA. All splice failure: (primer pair: GV3255/GV3256) amplifies a PCR product only in the incorrectly spliced RNA. Data show the mean number of calculated copies/PCR and standard (±SE) of four independent experiments (n = 4) performed in triplicate. Statistical significances are shown. n.s., nonsignificant indicates P > 0.05, paired t test.
Tables
- Table 1.
Comparison of recombineering efficiencies of exogenous/synthetic intronization (ESI) mutagenesis and counterselection.
Artificial intronization (ESI) Counterselection Mutations Insertion (PCR) Sequencing Total % Cassette insertion (PCR) Rescue (PCR) Sequencing Total % GTSE1 (SXIP) 32/50 7/10 44.8% 5/8 7/50 7/7 8.8% GTSE1 (T165A) 30/50 10/12 50.0% 5/8 44/50 10/10 55.0% CHC (C) 22/100 6/7 17.1% 6/8 41/50 10/10 61.5% Average 48% 80% 37.3% 67% 61% 100% 41.8%
Supplemental Data 1.
Exogenous/synthetic intronization bacterial artificial chromosome mutagenesis.[LSA-2020-00836_Supplemental_Data_1.docx]
Table S2 Oligos for RT-PCR splicing analysis.
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