FIGURE LEGENDS
Figure 1: Overview of hnRNPA1 recruiting molecules (AROs).(A) Each ARO consists of two parts, a 20 bp hnRNPA1 recruiting loop derived from HIV exon splicing silencer 3, which recruits hnRNPA1 by binding to RNA binding domain RRM1, and an antisense targeting domain consisting of 20-25 base pairs of RNA complementary to a sequence on a target pre-mRNA. (B) The hypothesized mechanism of AROs. 1. In the absence of AROs splicing occurs normally. 2. The binding of an ARO to its pre-mRNA target recruits hnRNPA1 and displaces SR proteins and spliceosome machinery (U2, U2AF65/35, and U1). 3. We hypothesize this causes repression of splicing resulting in exon skipping or intron inclusion. This would cause frameshifts in the mRNA leading to the presence of premature termination codons (PTCs) in the reading frame upstream of exonic junction complexes (EJCs). UPF1 would then recognize these sites as mis-spliced mRNA and degrade the RNA by non-sense mediated decay.
Figure 2: Plasmid expressed AROs knock down KRT14 in human keratinocytes (HaCat cells). (A) Visual representation of each ARO and controls. 1. scrOligo-ARL is a scrambled targeting oligo with an intact hnRNPA1 recruiting loop (ARL). 2. Oligo is a targeting oligo with homology to Exon 7 of KRT14. 3. ARL-Oligo is the targeting oligo with an ARL on the 5’ end of the construct relative to the targeting domain. 4. Oligo-ARL is the targeting oligo with an ARL on the 3’ end of the construct relative to the targeting domain. 5. ARL-Oligo-ARL is a construct with ARLs attached to both the 5’ and 3’ ends of the targeting domain. (B) Fold change of KRT14 transcript expression relative to a carrier only lipofectamine control RT-qPCR. Cells were lipofected with plasmid for 48 hours and then harvested for RNA extraction. Green bars indicate plasmid experiments while blue bars indicate experiments with siRNA designed to knock down hnRNPA1. Three replicates were performed. Error bars show standard deviation between replicates. A Student’s t test was used to determine significance.
Figure 3: Plasmid expressed AROs targeting exon 14 knock down TBK1 RNA expression in human keratinocytes (HaCat cells). All AROs were expressed with ARLs attached to the 3’ and 5’ ends of the targeting oligo. (A) Location of targeting oligo homology to exon 14 of TBK1. (B) Fold change of TBK1 transcript expression relative to a carrier only lipofectamine control RT-qPCR. Cells were lipofected with plasmid for 48 hours and then harvested for RNA extraction. Three replicates were performed. Error bars show standard deviation between replicates. A Student’s t-test was used to determine significance.
Figure 4: Synthesized single-strand RNA oligonucleotides knock down KRT14 expression across two cell types. (A) Synthesized ssRNAs with phosphorothioate bonds on the first two and last two bases administered via lipofection to HaCat cells at varying concentrations. ARL-Oligo-ARL refers to a ssRNA with two hnRNPA1 recruiting loops on either side of the targeting domain. ARL-Oligo is composed of a single hnRNPA1 recruiting loop fused to the 5’ end of an RNA targeting sequence. A previously validated siRNA against KRT14 was used as a positive control at each concentration. Cells were harvested 48 hours post lipofection and fold change was determined relative to a lipofectamine carrier only control. (B) The single ARL-Oligo ssRNA and siRNA targeting KRT14 were lipofected into SCC9 cells. As previously, cells were harvested 48 hours post lipofection and fold change was determined relative to a lipofectamine carrier only control. Three replicates were performed in each experiment. Error bars show standard deviation between replicates. A Student’s t-test was used to determine significance.
Figure 5: AROs show a functional phenotype in a cancer invasion model. (A) As previously, siRNA targeting KRT14 and an ssRNA ARO with a single hnRNPA1 recruiting loop fused to the 5’ end of a KRT14 targeting oligo were administered at 100 nM via lipofection in SCC9 cells and grown for 48 hours. 20,000 cells were seeded inside a matrigel-coated transwell for a Boyden chamber invasion assay and allowed to migrate through the pores to the other side of the membrane for 24 hours. Cells were then stained with crystal violet and invading cells were imaged and counted. Two replicates were preformed of this experiment. Error bars show standard deviation between replicates. A Student’s t test was used to determine significance. (B)Representative images of crystal violet stained invading cells in each condition. (C) Protein abundance of KRT14 in cells lipofected with either ARO (ssRNA ARL-Oligo) or anti-KRT14 siRNA at a final concentration of 100 nM. Each condition was performed in triplicate and total protein was harvested 48 hours after lipofection. Western blots were preformed using KRT14 mouse primary antibody and an anti-mouse HRP conjugated secondary antibody, and total protein load using a stain free gel was used as a loading control for normalization.