Fig 2: Defining the nature of RNA exosome targets. (A) Definition of gene/RNA classes. The left schematic shows the set of rules used for defining whether changes in RNA levels are mostly driven by transcription, exosome degradation or both. Only transcripts with an absolute log2 fold change of > 0.5 between ESCs and EBd3 were analyzed. Bar plots to the right show the numbers of genes/RNAs in each class. Note that bar colors were used consistently in Figure 5 to indicate the classes, only the ‘other’ class was not further analyzed. (B) Left panel: fractions of coding and non-coding genes/RNAs from the categories in (A). Right-panel: fractions of different types of non-coding genes/RNAs from the left panel. (C) RNAseq log2 fold changes between RRP40 depleted and control ESC samples along each exon (white) and intron (gray) of pre-mRNAs from the classes ‘mainly exosome degradation’ and ‘combination transcription and exosome degradation’ from (A). The number of transcripts analyzed (N) is indicated above the panel. (D) UCSC genome browser (60) examples of genes whose transcripts are downregulated in ESC (top, Prph locus) or EBd3 (bottom, Cish locus) samples, mainly due to exosome degradation. Genome browser tracks show, from top to bottom, signal intensities of CAGE, RNAseq and PROseq samples for each experimental condition on the relevant strand (as indicated by color). Numbers to the left indicate the scale for the respective data type. Bottom track shows GENCODE annotation. Arrow indicates the direction of transcription. The CAGE tracks show pooled data from the two replicates and the RNAseq and PROseq tracks show one of the replicates. (E) Distribution of RNAseq (top) and PROseq (bottom) average signals (normalized log2(TPM)) for RNAs in the classes from panel (A). P-values indicate results from Mann–Whitney two-sided tests between distributions. (F) Distributions of absolute log2(EBd3 versus ESC fold change) for RNAs in the classes from panel (A). P-values indicate results from Mann–Whitney two-sided tests between distributions. (G) Left panel: Distributions of gene lengths (log2(bp), including introns) (left). Right panel: exon count distributions for transcripts in the classes from panel (A), log2 scaled. P-values indicate results from Mann–Whitney two-sided tests between distributions.

Fig 3: Effects of RRP40 depletion in ESCs and EBs. (A) Schematic representation of the experimental procedure and data collection. Scrambled control or RRP40-specific shRNAs were introduced using lentiviral vectors into ESCs, after which cells were differentiated into EB for 3 days. From each cell state and shRNA condition, CAGE, RNAseq and PROseq data were collected. The scale bar on the lower right corner of the images represents 100 µm (ESC) and 200 µm (EBd3). (B) Western blotting analysis of RRP40 levels in ESCs treated with the indicated shRNAs and differentiated for 0 (left) or 3 (right) days. Tubulin was used as a loading control. (C) Distributions (y-axis) of log2 fold changes (x-axis) between RNAseq (green) or PROseq (blue) values from RRP40-depleted versus control ESC samples. The RNA types measured and their numbers are indicated above each panel. The average of all replicates is shown. (D) As in (C) but for the EBd3 samples.

Fig 4: Model of HBS1LV3-exosome interactions. HBS1LV3 helix (aa 555–576) docks into the surface of RRP43. (A) Modeled human HBS1LV3 helices docked into the exosome core (PDB code: 2NN6, (39)) are shown in blue. The RRP43 protein is shown in green. The remainder of the human exosome complex is colored grey. The 10 best predictions are shown, with 5 predictions docking into the same position on the surface of the RRP43 exosome subunit. The highest ranking prediction is colored red. Bottom: secondary structure prediction of HBS1LV3 fragment. (B) A magnified view of interactions. (C) R559, F563 and L567 HBS1LV3 amino acids are crucial for interactions with the exosome complex. Total cell extracts were prepared from HEK293 cells transiently transfected with vectors expressing either EGFP-tagged HBS1LV3 (WT) or HBS1LV3 MUT (R559A, F563A, L567A) proteins. Co-IPs were performed using GFP trap resin. The complete cell extracts (INPUT) and precipitated proteins (IP) were separated by SDS-PAGE and analyzed by Western blotting. a-HBS1L, a-RRP40 and a-SKI2W antibodies were used to visualize precipitated proteins. a-GAPDH antibodies were utilized as a loading control.

Fig 5: Homozygosity mapping, exome sequencing, EXOSC8 mutation analysis and immunoblotting for EXOSC8.(a) Genome-Wide Human SNP Array 6.0 (Affymetrix) was performed in six affected (P1-V:2, P1-V:10, P1-V:20, P1-V:27, P1-V:29, P1-VI:3) and one unaffected family members (P1-IV:14). Homozygosity mapping indicated two regions of homozygosity on chromosome 13 spanning from 36214563, bp to 37767059, bp (rs7327540 to rs11147637) and 43243791, bp to 44640995, bp (rs9533208 to rs9567354). (b) Exome sequencing detected 2,294 and 2,274 rare/novel single nucleotide variants (SNV) of which 184 were shared homozygous variants in patients P1-V:10 and P1-V:29; seven were protein altering, one of which was in the region of homozygosity in EXOSC8. SNV-Varscan parameters (*) minimum total coverage=fivefold, minimum variant coverage =onefold, minimum quality>10; Indel-Dindel output filter minimum variant coverage =4. (a) Variants with position within targets (Illumina Truseq 62 Mb) ±500 bp, seen on both (forward and reverse) strands and (SBVs only) variant allele frequency >24%. (b) Variants that match 1,000 Genomes (Feb2012) and/or NHLBI 6500 exomes and/or In-house 334 exomes with MAF>0.01. (c) Confirmatory Sanger Sequencing indicated the presence of the homozygous c.815 G>C, p.Ser272Thr mutation in an affected patient (A) and heterozygously in her mother (U). This rare variant is listed as rs36027220 with a minor allele frequency of <0.01 (ref database) and was present also in pedigree 2. In pedigree 3 another homozygous mutation c.5C>T, p.Ala2Val has been identified (control - upper chromatogram, patient - lower chromatogram). (d) Immunoblotting showing EXOSC8 protein in control myoblasts (C. M.) and in myoblasts of P1-V:10 (P. M.); control fibroblasts (C. F.) and in fibroblasts of P3-II:1 (P. F.) using ß-Actin or GAPDH as a loading control. (e) Both mutations alter conserved amino acids. (f) Crystal structure of the human exosome complex. The cap proteins are indicated in green (EXOSC3 light green) and the core proteins in grey. EXOSC8 highlighted in pink and the mutated residues (Ser 272, Ala2) are marked as black (arrowhead).