Journal: Science Advances

Article Title: Spliceosome buffers cryptic genetic variation to enforce phenotypic robustness in Arabidopsis

doi: 10.1126/sciadv.aed7513

Figure Lengend Snippet: ( A ) Embryonic development in transgenic plants expressing either the genomic ( gHIKI1 L er ) or CDS ( cHIKI1 L er ) of HIKI1 L er in skip-1 +/− plants. ( B ) Segregation of skip-1 in (A) T2 population. Detailed chi-square values were in table S2. ( C ) Alternative splicing analysis of HIKI1 Col-0 , HIKI1 L er , and SNP1123 point mutants in SKIP and skip-1 backgrounds. RT-PCR primer positions were indicated. Sanger sequencing revealed three splice isoforms (α, β, and γ). ( D ) Quantification of splice-isoform abundance. Grayscale intensities from (C) were used to calculate each variant’s percentage of the total signal. ( E ) RNA-immunoprecipitation quantitative polymerase chain reaction (RIP-qPCR) showing direct binding of SKIP to HIKI1 pre-mRNA. Immunoprecipitated product was verified by Sanger sequencing. APX3 , negative control . Experiments were repeated three times with similar results. IgG, immunoglobulin G. ( F ) RIP-qPCR quantified SKIP occupancy along HIKI1 pre-mRNA. ( G ) Left: RIP-qPCR quantified SKIP occupancy along HIKI1 Col-0 and HIKI1 L er pre-mRNA at E site (F) in Col-0 and L er accessions. Right: RIP-qPCR quantified SKIP occupancy along HIKI1 Col-0 and HIKI1 L er pre-mRNA at E site in skip-1 hiki1-1 protoplasts transiently expressing HIKI1 Col-0 and HIKI1 L er . ( H ) Seed development in skip-1 +/− plants expressing splice variants: α HIKI1 L er , β HIKI1 L er , and γ HIKI1 L er , respectively. The spliced transcripts of HIK1 correspond to the three splicing isoforms shown in (C). ( I ) Segregation of skip-1 in (H) T2 population. Detailed chi-square values were in table S2. Data in (B), (D), (F), (G), and (I) represented means ± SD. Data were from three [(B), (F), (G), and (I)] or five (D) biological replicates. In (A) and (H), percentage, aborted seeds/total seeds (summary of three biological replicates); white arrow, aborted seed; scale bars, 0.5 mm.

Article Snippet: Immunoprecipitation was performed overnight at 4°C with 2 μl of green fluorescent protein antibody (Abcam, ab290) or 10 μl of rabbit control immunoglobulin G (ABclonal, AC005) followed by 2-hour capture on 25 μl of rProtein A/G magnetic beads (Thermo Fisher Scientific, 26162).

Techniques: Transgenic Assay, Expressing, Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, Sequencing, RNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Immunoprecipitation, Negative Control