HEK293 conformation-locking mutagenesis MPRA of RNA switches
A systematic search for RNA structural switches across the human transcriptomeA second lentiviral MPRA library tested four designed mutations per candidate RNA switch, with two perturbations favoring each of two predicted mutually exclusive RNA conformations. The same two-replicate, eight-bin HEK293 eGFP:mCherry sorting and genomic-DNA/RNA sequencing design quantified conformation-dependent reporter activity.
Processed tables are specific to each experiment. Column names, units, measurements, and table structure are not standardized across the database. Check this experiment’s column definitions and quality-control notes before comparing or combining data.
Perturbation & assay details
Basal / Untreated
Designed stem-strengthening and stem-weakening substitutions were introduced to shift the RNA ensemble toward conformation 1 or 2. Constructs were cloned into the same dual eGFP–mCherry 3′UTR lentiviral reporter, transduced at low MOI, sorted into eight equal eGFP:mCherry bins, and quantified from paired genomic-DNA and RNA libraries.
Processed data
50 rows per page. Click a cell to inspect its full value.
Visible columns (73 of 73)
| Row | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 3 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 4 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 5 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 6 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 7 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 8 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 9 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 10 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 11 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 12 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 13 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 14 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 15 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 16 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 17 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 18 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 19 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 20 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 21 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 22 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 23 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 24 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 25 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 26 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 27 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 28 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 29 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 30 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 31 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 32 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 33 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 34 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 35 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 36 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 37 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 38 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 39 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 40 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 41 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 42 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 43 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 44 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 45 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 46 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 47 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 48 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 49 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 50 |
Filters apply to this table only. The CSV download contains the complete processed table; filtered rows are available through the API.
Column dictionary · 73 definitions
- element_id
- Source RNA switch fragment identifier.
- candidate_id
- Unique candidate identifier combining element_id and the source SwitchFinder score; disambiguates repeated fragment names with distinct predictions.
- construct_id
- Unique construct identifier combining candidate_id and the mutation perturbation.
- transcript_id
- Ensembl transcript identifier parsed from element_id.
- fragment_index
- Overlapping 186-nt 3′UTR fragment index parsed from element_id.
- perturbation
- Source mutation design: strengthen or weaken conformation 1 or 2.
- mutation_target_conformation
- Predicted conformation favored by the perturbation, 1 or 2.
- mutation_mode
- Mutation strategy: strengthen or weaken the target conformation-specific stem.
- sequence
- DNA sequence of the designed mutant tested in the reporter.
- sequence_length_bp
- Length of the tested sequence in base pairs.
- gc_fraction
- Fraction of sequence bases that are G or C.
- switchfinder_score
- Source SwitchFinder RNA-switch score.
- is_published_high_confidence_switch
- Source RNA switch flag; true identifies the 245 switches reported as final high-confidence switches.
- candidate_variant_count
- Number of perturbation constructs in the retained candidate group; four expected.
- candidate_complete_qc
- True when all four expected perturbations for this candidate passed QC.
- dna_r1_b1_count
- Source genomic-DNA read-count value for biological replicate 1, FACS expression bin 1.
- dna_r1_b2_count
- Source genomic-DNA read-count value for biological replicate 1, FACS expression bin 2.
- dna_r1_b3_count
- Source genomic-DNA read-count value for biological replicate 1, FACS expression bin 3.
- dna_r1_b4_count
- Source genomic-DNA read-count value for biological replicate 1, FACS expression bin 4.
- dna_r1_b5_count
- Source genomic-DNA read-count value for biological replicate 1, FACS expression bin 5.
- dna_r1_b6_count
- Source genomic-DNA read-count value for biological replicate 1, FACS expression bin 6.
- dna_r1_b7_count
- Source genomic-DNA read-count value for biological replicate 1, FACS expression bin 7.
- dna_r1_b8_count
- Source genomic-DNA read-count value for biological replicate 1, FACS expression bin 8.
- dna_r2_b1_count
- Source genomic-DNA read-count value for biological replicate 2, FACS expression bin 1.
- dna_r2_b2_count
- Source genomic-DNA read-count value for biological replicate 2, FACS expression bin 2.
- dna_r2_b3_count
- Source genomic-DNA read-count value for biological replicate 2, FACS expression bin 3.
- dna_r2_b4_count
- Source genomic-DNA read-count value for biological replicate 2, FACS expression bin 4.
- dna_r2_b5_count
- Source genomic-DNA read-count value for biological replicate 2, FACS expression bin 5.
- dna_r2_b6_count
- Source genomic-DNA read-count value for biological replicate 2, FACS expression bin 6.
- dna_r2_b7_count
- Source genomic-DNA read-count value for biological replicate 2, FACS expression bin 7.
- dna_r2_b8_count
- Source genomic-DNA read-count value for biological replicate 2, FACS expression bin 8.
- rna_r1_b1_count
- Source RNA read-count value for biological replicate 1, FACS expression bin 1.
- rna_r1_b2_count
- Source RNA read-count value for biological replicate 1, FACS expression bin 2.
- rna_r1_b3_count
- Source RNA read-count value for biological replicate 1, FACS expression bin 3.
- rna_r1_b4_count
- Source RNA read-count value for biological replicate 1, FACS expression bin 4.
- rna_r1_b5_count
- Source RNA read-count value for biological replicate 1, FACS expression bin 5.
- rna_r1_b6_count
- Source RNA read-count value for biological replicate 1, FACS expression bin 6.
- rna_r1_b7_count
- Source RNA read-count value for biological replicate 1, FACS expression bin 7.
- rna_r1_b8_count
- Source RNA read-count value for biological replicate 1, FACS expression bin 8.
- rna_r2_b1_count
- Source RNA read-count value for biological replicate 2, FACS expression bin 1.
- rna_r2_b2_count
- Source RNA read-count value for biological replicate 2, FACS expression bin 2.
- rna_r2_b3_count
- Source RNA read-count value for biological replicate 2, FACS expression bin 3.
- rna_r2_b4_count
- Source RNA read-count value for biological replicate 2, FACS expression bin 4.
- rna_r2_b5_count
- Source RNA read-count value for biological replicate 2, FACS expression bin 5.
- rna_r2_b6_count
- Source RNA read-count value for biological replicate 2, FACS expression bin 6.
- rna_r2_b7_count
- Source RNA read-count value for biological replicate 2, FACS expression bin 7.
- rna_r2_b8_count
- Source RNA read-count value for biological replicate 2, FACS expression bin 8.
- gdna_total_rep1
- Sum of source genomic-DNA values across bins 1–8 for replicate 1, on the source count scale.
- rna_total_rep1
- Sum of source RNA values across bins 1–8 for replicate 1, on the source count scale.
- normalized_gdna_total_rep1
- Median-of-ratios normalized genomic-DNA total for replicate 1.
- normalized_rna_total_rep1
- Median-of-ratios normalized RNA total for replicate 1.
- activity_log2_rna_dna_rep1
- Log2 of normalized RNA total divided by normalized genomic-DNA total for replicate 1, using a 0.5 pseudocount.
- mean_rna_expression_bin_rep1
- RNA-count-weighted mean FACS bin number (1–8) for replicate 1.
- dna_adjusted_expression_bin_rep1
- Mean FACS bin number weighted by per-bin normalized RNA/genomic-DNA enrichment for replicate 1, with 0.5 pseudocounts.
- gdna_total_rep2
- Sum of source genomic-DNA values across bins 1–8 for replicate 2, on the source count scale.
- rna_total_rep2
- Sum of source RNA values across bins 1–8 for replicate 2, on the source count scale.
- normalized_gdna_total_rep2
- Median-of-ratios normalized genomic-DNA total for replicate 2.
- normalized_rna_total_rep2
- Median-of-ratios normalized RNA total for replicate 2.
- activity_log2_rna_dna_rep2
- Log2 of normalized RNA total divided by normalized genomic-DNA total for replicate 2, using a 0.5 pseudocount.
- mean_rna_expression_bin_rep2
- RNA-count-weighted mean FACS bin number (1–8) for replicate 2.
- dna_adjusted_expression_bin_rep2
- Mean FACS bin number weighted by per-bin normalized RNA/genomic-DNA enrichment for replicate 2, with 0.5 pseudocounts.
- activity_log2_rna_dna
- Mean of the two replicate log2 RNA/genomic-DNA activity values.
- activity_log2_rna_dna_sd
- Sample standard deviation of the two replicate log2 RNA/genomic-DNA activity values.
- activity_log2_rna_dna_rep2_minus_rep1
- Replicate 2 minus replicate 1 log2 RNA/genomic-DNA activity.
- mean_rna_expression_bin
- Mean of the two RNA-weighted mean FACS-bin scores.
- dna_adjusted_expression_bin
- Mean of the two DNA-adjusted expression-bin scores.
- expression_bin_score_rep2_minus_rep1
- Replicate 2 minus replicate 1 RNA-weighted mean-bin score.
- replicate_bin_profile_pearson_r
- Pearson correlation between the two replicate per-bin RNA/genomic-DNA enrichment profiles.
- candidate_conformation_1_mean_activity
- Mean activity_log2_rna_dna across the two retained perturbations favoring conformation 1 for this candidate.
- candidate_conformation_2_mean_activity
- Mean activity_log2_rna_dna across the two retained perturbations favoring conformation 2 for this candidate.
- candidate_conformation_2_minus_conformation_1_activity
- Candidate-level conformation 2 mean activity minus conformation 1 mean activity; repeated on each variant row.
- variant_activity_vs_target_conformation_mean
- This variant's activity minus the mean activity of the other perturbation targeting the same conformation.
- variant_activity_vs_opposite_conformation_mean
- This variant's activity minus the mean activity of the two perturbations targeting the opposite conformation.
Quality control
Author workflow retained: approximately 20% low-MOI lentiviral infection; eight equal 12.5% FACS bins; two biological replicates; cutadapt trimming, UMI handling, BWA-MEM mapping, featureCounts quantification, median-of-ratios normalization, and replicate/candidate ranking. Additional package QC required valid A/C/G/T sequence and at least 100 source values summed across all eight bins for both RNA and genomic DNA in each replicate. Only complete four-perturbation candidate groups were retained. 5116/5128 construct rows (1279 candidate score groups) passed.
Curation notes
Source: GSE266058 mutagenesis_screen.tsv.gz (publisher Supplementary Data S5); the complete source matrix is preserved in raw_data. The public table has four perturbation rows per candidate score group and no individual barcode identifiers. candidate_id combines RNA switch name and SwitchFinder score because some fragment names occur with more than one prediction. The source RNA switch TRUE/FALSE flag is retained as is_published_high_confidence_switch; after QC, 244 high-confidence candidate groups remain. Source bin values are retained; derived activity and bin scores use the same median-of-ratios and pseudocount calculation documented for the functional screen. A positive candidate_conformation_2_minus_conformation_1_activity means the perturbations favoring conformation 2 had higher reporter RNA/DNA activity on average.