The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations

Abstract

Cancer is a disease of the genome caused by oncogene activation and tumor suppressor gene inhibition. Deep sequencing studies including large consortia such as TCGA and ICGC identified numerous tumor-specific mutations not only in protein-coding sequences but also in non-coding sequences. Although 98% of the genome is not translated into proteins, most studies have neglected the information hidden in this "dark matter" of the genome. Malignancy-driving mutations can occur in all genetic elements outside the coding region, namely in enhancer, silencer, insulator, and promoter as well as in 5'-UTR and 3'-UTR Intron or splice site mutations can alter the splicing pattern. Moreover, cancer genomes contain mutations within non-coding RNA, such as microRNA, lncRNA, and lincRNA A synonymous mutation changes the coding region in the DNA and RNA but not the protein sequence. Importantly, oncogenes such as TERT or miR-21 as well as tumor suppressor genes such as TP53/p53, APC, BRCA1, or RB1 can be affected by these alterations. In summary, coding-independent mutations can affect gene regulation from transcription, splicing, mRNA stability to translation, and hence, this largely neglected area needs functional studies to elucidate the mechanisms underlying tumorigenesis. This review will focus on the important role and novel mechanisms of these non-coding or allegedly silent mutations in tumorigenesis.

Keywords: alternative polyadenylation; enhancer; mutation; non‐coding RNA; synonymous mutation.

Figure 1. Schematic depiction of mutations within the 5′‐ and 3′‐UTR

Mutations can alter the secondary structure of the 5′‐ or 3′‐UTR or occur in RNA binding protein (RBP) binding sites, upstream ORFs (uORF), internal ribosome entry sites (IRES; ITAF: IRES trans‐acting factor), start codons of open reading frames (ORF), microRNA binding sites, or polyadenylation signals (polyA). These alterations can affect translation efficiency, mRNA stability, ORF length, or RBP interaction as well as cause alternative cleavage and polyadenylation (APA). Prominent examples of genes involved in tumorigenesis (green: induced, red: decreased) that exhibit mutations (red star) in such elements are illustrated.

Figure 2. Functional mechanisms caused by synonymous mutations

(A) Synonymous mutations can affect mRNA splicing. Of the possible splice events, an example of exon skipping by deletion of an exonic splicing enhancer (ESE) is shown (red). (B) Synonymous mutations can alter mRNA stability by modifying the secondary mRNA structure and lead to either translation (green) or mRNA degradation (red). (C) Protein abundance can be altered by either creating (red) or abrogating (green) a miRNA binding site in the mRNA. (D) Synonymous mutations can affect translation at different stages: (Da) translation initiation is favored by a less complex mRNA secondary structure (green) and hindered by a more stable secondary structure around the start codon (red). (Db) Codon choice and tRNA availability can either increase (green) or decrease (red) translational elongation speed. (Dc) The removal or creation of a ribosomal pause site can alter the protein conformation and structure. A ribosomal pause site in between two domains allows the translated domains to fold independently (green). The removal of a ribosomal pause site allows the cooperate folding of the two domains (red).

Figure 3. Sites and effects of intronic splicing mutations in cancer

(A) Mutations in introns affecting splicing. a) Mutations in the intron can destroy a consensus splice donor or acceptor site (SpS) at the intron boundaries. b) Intronic mutations can create a novel splice donor or acceptor site (SpS). c) Mutations in introns can either create or destroy a branch point (BP). d) Mutations in splicing regulatory elements (SRE) can cause the formation or deletion of an intronic splicing silencer (ISS) or enhancer (ISE). (B) Products of splicing mutations. Depending on the presence of cryptic splice sites in the vicinity of the aforementioned mutations, the mutations can result in all product types of alternative splicing.