Supplementary Figure 13: Impact of microRNAs on mRNA stability in mESCs.

(a) Schematic representation of microRNA (miRNA)-mediated gene regulation. Target site-types with increasing (top to bottom) repressive function are shown. (b) Abundance of microRNAs (miRNAs) in mESCs. Relative abundance was determined by small RNA sequencing. Members of the miR-291-3p/294-3p/295-3p/302-3p-family are highlighted in red. The miRNAs miR-292a-3p and miR-291b-3p (highlighted in rose) share a 6mer seed with the miR-291-3p/294-3p/295-3p/302-3p-family. Together, these miRNAs (referred to as “miR-291a-family”) represent more than half of all miRNAs in mESCs. (c) Schematic representation of the Xpo5 gene locus in the mouse genome. Zoom-in shows a region in the first coding exon of Xpo5, which was targeted by CRISPR/Cas9 genome editing. Disruptive frameshift mutations in three independent xpo5^ko clones are shown. (d) Western blot analysis for Exportin-5 (Xpo5) and Actin in wild-type (wt) mESCs and three independent xpo5^ko clones. n/a, alternative clones not used in this study. (e) Northern hybridization assay for miR-290-5p and U6 snRNA in wild-type mESCs and three independent xpo5^ko clones. n/a, alternative clones not used in this study. (f) MicroRNA-abundance determined by spike-in-controlled small RNA sequencing in xpo5^ko mESCs relative to wild-type cells. Ratio for all miRNAs (black), miR-291a-family (red), miR-290a-family (rose), miR-293-family (light grey) and miR-291-family (dark grey) are shown. Data represent mean (center line) ± SD (whiskers) of three independent xpo5^ko clones shown in (c) and (d). (g) Abundance of miR-families in wildtype mESCs as determined by small RNA sequencing. Highlighted are the most abundant miR-families, which are all transcribed from the miR-290-295 cluster: Members of the miR-291-3p/294-3p/295-3p/302-3p and miR-292a-3p/467a-5p family (“miR-291a”, in red), miR-290a-5p/292a-5p-family (“miR-290a”, in rose), miR-293-3p-family (“miR-293”, in light grey) and miR-291-5p-family (“miR-291”, in dark grey). (h) Cumulative distribution of ranked mRNA stabilities. Plotted are distributions for transcripts harboring no predicted target sites for any miRNA belonging to the ten most highly expressed miR-families (black, n=2188); at least one predicted target site for miRNAs belonging to the miR-291a-family (red, n=1450); at least one predicted target site for miRNAs belonging to the miR-290a-family, but no site for miR-291a-family miRNAs (rose, n=1326); at least one predicted target site for miR-293, but no site for miR-291a-family or miR-290a-family miRNAs (light grey, n=117); at least one predicted target site for miR-291 miRNAs, but no predicted sites for miR-291a-family, miR-290a-family or miR-293 family miRNAs (dark grey, n=708). P-value was determined by KS-test. (i) Cumulative distribution of mRNA stability changes in xpo5^ko relative to wt mESCs. Plotted are distributions for transcripts that have predicted miRNA target sites as described in (b). P-value was determined by KS-test. N.s., not significant (p>0.05).