IGV view (Integrative Genomics Viewer) showing the enrichment of AGO-CLIP sequencing reads in miR-4728-3p transfected vs non-transfected control around the IS target site on the 3′UTR of the Ubiquitin carboxyl-terminal hydrolase 1 (USP1) gene.

<p>The positions of the 7- and 6-mer IS target sites are highlighted below the USP1 gene.</p

IS targeting is not restricted to miR-4728-3p.

<p>SylArray enrichment landscape from a microarray experiment of miR-30a overexpression in HCT116 cells. Data generated by Baraniskin <i>et al.</i> was analyzed by SylArray. Word designations and graph details as in Fig. 1a, with CS and IS referring to corresponding miR-30a positions.</p

miR-4728-3p IS regulates ESR1.

<p><b>A.</b> qRT-PCR analysis of ESR1 and HER2 transcripts and miR-4728-3p among a panel of 38 breast cancer tumors (19 HER2+, 19 HER2-). Calibrated Normalized Relative Quantity (CNRQ) of miR-4728-3p (left) and HER2 (right) is plotted against expression levels of ESR1. Tumors classified as HER2+ by ISH are shown in red, HER2- in grey. Expression was normalized to a panel of reference genes. For details see text and material and methods. <b>B.</b> Luciferase assay in BT-474 with ESR1 3′UTR constructs carrying either wild type target site of miR-4728-3p internal seed (WT) or mutated internal seed site (MUT). Firefly luciferase activity was normalized against Renilla luciferase. Reporter activity is given as % of WT in respective experiment. Repression of WT ESR1 construct by endogenous miR-4728-3p (left) is alleviated by an antisense oligo (AS) against endogenous miRNA (right) but not by a non-targeting control (middle). <b>C.</b> Western blot (left) and protein quantification (right) of ESR1 in MCF7. The two main isoforms of ESR1 (47 and 66 kDa), plotted as percentage of control signal of matching size, are down regulated upon transfection of miR-4728-3p mimics. Levels of HER2, (p)MAPK and (p)AKT remain largely unchanged. <b>D.</b> MCF7 cells were transfected with indicated concentrations of miR-4728-3p mimic. ESR1 levels show a concentration-dependent down-regulation that is most pronounced at highest tested concentration (25 nM). <b>E.</b> Western blot (left) and protein quantification (right) of ESR1 in BT474. ESR1 is up regulated when blocking endogenous miR-4728-3p with AS-oligonucleotides, while pMAPK and pAKT remain largely unchanged. <b>F.</b> Western blot (left) and protein quantification (right) of ESR1 in HCC1954 cells. ESR1 isoform of 47 kDa is up regulated under miR-4728-3p blocking. The main 66 kDa isoform is not detectable in this ER- cell line. Signals were quantified with ImageJ and normalized to total protein by Coomassie stain. Tubulin was used as a loading control. Asterisks denote p-values of <0.05 (*), and <0.005 (**) in Student′s t-test.</p

The main isoform of miR-4728-3p is 24-26nt long and interacts with its targets through an internal seed.

<p><b>A.</b> SylArray enrichment landscape plots for 6-, 7- and 8-mer words (from top to bottom) for ranked genes from a microarray experiment of miR-4728-3p overexpression in MCF 10A. 3′ UTRs are sorted on the x-axis from most down (left) to up regulated (right). Significance of the words is given as log-transformed P-values, where over- and underrepresentation are shown on the positive and negative y axis, respectively. Highlighted are targets corresponding to canonical seed (CS, red), CS shifted by one base towards the miRNA'3′ end (CS+1, green), internal seed (IS, blue) and IS +1 (purple). CS+1 is below significance cut-off and it is not highlighted for 8-mer. <b>B.</b> Northern blot of total RNA from cells A) untransfected and B) transfected with miR-4728-3p mimics with a radiolabeled miR-4728-3p probe. Control lane shows signal from synthetic miR-4728-3p RNA and the ethidium bromide staining in the lower panel shows tRNA bands as loading control. <b>C.</b> Alignment of small RNA sequencing reads to miR-4728-3p genomic context. Positions of CS, CS+1, IS, and IS+1 are highlighted in color as in panel A above the alignment. Sequencing reads of mir-4728 in cell lines with endogenous expression (BT-474, JIMT1 and SKBR3), mimic-transfected MCF 10A (Mimic) and tumors are given to the right as percentages of total miR-4728-3p reads. <b>D.</b> Empirical cumulative distribution functions (ECDF) show effectiveness of CS and IS target sites. mRNA abundance after miRNA transfection in MCF 10A was monitored with microarrays. Distributions of changes for 3′ UTRs of mRNAs containing CS, IS, both, or neither are colored as denoted on the right. Each class contains the seed and the respective shifted seed (+1). <b>E.</b> Conservation plot. Top 250 down regulated genes from a microarray experiment of miR-4728-3p overexpression were filtered for IS and CS target sites respectively. Target sequence context of 10 bases on either side of the seeds was extracted and analyzed with multiple sequence alignment. The IS conservation plot shows conservation of the target site but not of surrounding nucleotides. <b>F.</b> Distribution of duplex energies of 3' UTRs containing IS target sites comparing genes down regulated by miR-4728-3p overexpression (t-statistics of <-4, TRUE) with unaffected genes (t-statistics ≥4, FALSE). Thermodynamic stability of hybrid formation between targets and miR-4728-3p was calculated using RNAduplex from the Vienna package. RNA duplex score is shown on the x-axis while target density is expressed on the y-axis. Folding stability is higher in a small number of regulated targets but otherwise similar to non-regulated targets, and only one motif with high duplex stability was part of a longer motif (10nt).</p