paSNVs disrupting cleavage/polyadenylation signals are depleted in the normal population.

(A) Bioinformatics workflow used to analyse the effect of paSNVs on pre-mRNA cleavage and polyadenylation.

(B) Top, effects of UP- and DOWN-paSNVs on the APARENT2 score (mean±SEM) as a function of their position with respect to annotated pre-mRNA cleavage sites (CSs). Bottom, combined distribution of AWTAAA-affecting paSNVs in both datasets.

(C) Box plot showing that paSNVs disrupting polyadenylation signals are significantly less frequent compared to control groups of events in normal population.

(D) paSNVs disrupting polyadenylation signals are enriched for singletons, consistent with purifying selection against such events in normal population.

Cancer somatic mutations tend to disrupt functional cleavage/polyadenylation signals.

(A) Bar plot showing enrichment of paSNVs disrupting polyadenylation signals among cancer somatic mutations.

(B) Bar plot showing enrichment of SNVs affecting AWTAAA sequences in 3’UTRs close to annotated cleavage sites (CSs) among cancer somatic mutations.

(C) Box plot showing that somatic mutations disrupt stronger cleavage/polyadenylation signals in cancer.

(D) paSNVs disrupting polyadenylation signals occurs in more evolutionary conserved regions in cancer (mean PhastCons score in 15-nt window centred at SNVs).

(E) Distribution of DOWN-paSNVs across cancer types in the Pan-Cancer Analysis of Whole Genomes (PCAWG) project.

Somatic cancer mutations often disrupt cleavage/polyadenylation signals in tumour suppressor genes.

(A) Stacked bar plot showing enrichment of SNVs disrupting polyadenylation signals (DOWN-paSNVs) in tumour suppressors in cancer.

(B-C) Overrepresentation of (B) tumour suppressors but not (C) oncogenes among genes with cancer somatic DOWN-paSNVs, as compared to genes with cancer somatic BG-paSNVs. Fractions of tumour suppressors and oncogenes are also shown for all genes and genes containing cancer somatic nonsense (premature stop codons), missense (altered amino acid residues) and synonymous (synonymous codons) mutations. Note that the enrichment of tumour suppressors is stronger for DOWN-paSNVs compared to nonsense mutations.

(C) Top 10 GO Biological Process terms significantly enriched in genes with cancer somatic DOWN-paSNVs. Note the enrichment of apoptosis- and cell death-related functions.

(D) GO Molecular Function terms significantly enriched in genes with cancer somatic DOWN-paSNVs.

Disruption of cleavage/polyadenylation signals in tumour suppressors, along with other damaging mutations, may facilitate cancer progression.

(A-B) Enrichment of different groups of cancer somatic SNVs in (A) tumour suppressors and (B) oncogenes calculated using DigDriver relative to genes not listed in Cancer Census (non-Census) and presented with 95% confidence intervals. Note that DOWN-paSNVs and nonsense mutations are enriched in tumour suppressors but not in oncogenes. In contrast, oncogenes are often affected by missense mutations, as expected.

(C) Cancer somatic DOWN-paSNVs co-occur in the same tumour with non-synonymous damaging SNVs, a group of somatic mutations defined in 20, more often than BG-paSNVs. Note that the co-occurrence is particularly high for tumour suppressors.

(D) The overall frequency of non-synonymous damaging SNVs is significantly higher in the DOWN-paSNV-containing group compared to the DOWN-paSNV-lacking group of tumour suppressor genes.

Somatic cancer DOWN-paSNVs are sufficient to downregulate tumour suppressor genes.

(A-B) Gene-specific expression differences between DOWN-paSNV-containing and wild-type samples (ΔLog2 of copy number variation-normalized FPKM values; see Materials and Methods) reveal a consistently negative effect of DOWN-paSNV on tumour suppressor mRNA abundance in colorectal cancers. Box plots are shown for (A) an aggregated set of qualifying tumour suppressors and (B) individual genes from this set. Outliers are omitted for clarity.

(C) Wild-type and mutated sequences of the XPA tumour suppressor gene cleavage/polyadenylation signal. The PAS hexamer is enclosed within a box.

(D) Top, XPA cleavage site read-through minigenes and corresponding primers used for RT-qPCR analyses. Bottom, RT-qPCR data showing stronger read-through (weaker polyadenylation) in the mutant minigene.

(E) Top, luciferase expression minigenes. Bottom, luciferase assay revealing that the cancer-specific PAS mutation dampens the expression of the reporter gene.