Journal: bioRxiv

Article Title: Dynamics and functional roles of splicing factor autoregulation

doi: 10.1101/2020.07.22.216887

Figure Lengend Snippet: ( A ) Western blot (Materials and Methods) shows that endogenous SF2 levels decrease with increasing expression of the ectopic copy. We induced SF2(gDNA) cells at different 4-epiTC/dox concentration for 24hrs. Antibody staining details as in . ( B ) Endogenous SRSF1 in HEK293 cells has a high level of transcription, comparable to the ectopic CMV promoter. We analyzed the gel band intensity with highest induction level using a Bio-Rad Chemi-Doc Image Lab 6.0 band analyzer.

Article Snippet: Gel bands were detected using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and analyzed using a Bio-Rad ChemiDoc Image Lab 6.0 band analyzer.

Techniques: Western Blot, Expressing, Concentration Assay, Staining

Journal: bioRxiv

Article Title: Dynamics and functional roles of splicing factor autoregulation

doi: 10.1101/2020.07.22.216887

Figure Lengend Snippet: Splicing negative autoregulation modulates its feedback strength in response to variable total substrate load. ( A ) Two possible outcomes in response to total substrate ‘load’. (Left) ‘robust feedback’ scheme: the total SF2 level (purple line) and its splicing pattern (green line) remain constant across a broad range of substrate levels. The amount of SF2 involved in negative feedback is independent of ‘load’ level. (Right) ‘adaptive feedback’ scheme: more SF2 is produced (purple and green curves) via weakening negative autoregulatory splicing (i.e. dashed negative arrow in the top grey box), as increased substrate ‘load’ titrates away available SF2 in the cell. ( B ) The inducible synthetic SF2 target (SynTarget, short as SynT) cell line contains H2B-Cerulean fused with the spliceable 3’UTR of SRSF1. This synthetic gene is expressed under a Tet-On CMV promoter and stably integrated at the Flp-In locus in a T-REx HEK293 cell line. ( C ) SynT is a splicing target of SF2. We used RT-PCR and gel-imaged 3 isoforms of SynT cells with 100 ng/ml dox (left lane) and of SynT cells with transiently transfected SF2(cDNA) plasmid in 100 ng/ml dox (right lane). We found that SF2 overexpression promotes the splicing of SynT, increasing the expression of short isoforms. ( D ) We induced SynT at different levels and quantified the concentration of SynT isoform 1 (top row) and the functional SF2 isoform 1 (bottom two rows) by RT-qPCR (see qPCR qualification in ). We found SF2 levels remained unchanged by expression from a single copy of SynT (middle column, by inducing the stably integrated SynTarget with 100ng/ml dox), but increased ∼50% when multiple SynT copies were induced in the same cell (right column, by transiently transfecting SynT plasmid with 100ng/ml dox). qPCR results were verified by normalizing to two house-keeping genes, GAPDH and SDHA, respectively. Purple solid lines are guides to the eye matching the scheme in (A). The data represents the exponential of logarithmic mean of normalized qPCR reads. Error bars represent the minimum and maximum values over 3-10 experimental replicates. ( E ) SF2 splice isoform pattern changes in response to increased total substrates. We quantified SF2 isoforms using RT-PCR and analyzed the gel band intensity by Bio-Rad ChemiDoc Image Lab 6.0 band analyzer. Two gel band examples are presented: one from HEK293 control (left), the other from transient multiple copies of SynTarget (right). Multiple copies of SynTarget trigger ∼ 30% more SF2 isoform 1 (i.e. functional unspliced isoform) through splicing. The data represents the median of gel band intensity percentage reads and error bars represent the standard deviation over 6-7 experimental replicates. Green solid lines are guides to the eye matching the scheme in (A). ( F ) The expression level of splicing factors positively correlates with their target expression across 53 human tissue types (gray dots), where 5 example tissues (uterus, lung, breast, stomach, liver) are labeled in distinct colors. The splicing factor RNA expression levels (TPM) were extracted from the GTEx database. The respective target genes are selected based on PO-STAR2, specifically, the top 2% in each CLIP database. The three splicing factors, SRSF1, hnRNPA1, and PTBP1 are all autoregulated via negative splicing feedback. ( G ) The RNA regulation proteins without negative splicing feedback do not show correlative patterns as in (F).

Article Snippet: Gel bands were detected using SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific) and analyzed using a Bio-Rad ChemiDoc Image Lab 6.0 band analyzer.

Techniques: Produced, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Over Expression, Expressing, Concentration Assay, Functional Assay, Quantitative RT-PCR, Standard Deviation, Labeling, RNA Expression