Streptococcus agalactiae o estreptococo beta hemolítico del grupo B (EGB)

Given that RNA is such a functional biomolecule in the cell, it comes as no surprise that there are many complex and rare genetic diseases where the disregulation of RNA, or its aberrant function, contributes to onset or pathogenesis of the disease. We now review these known examples, covering first many of the examples where disruption of conserved sequence content is the causative agent. We then cover examples (some known, some hypothesized) where RNA structure disruption is the causative agent.

1.9.1 Many known examples of RNA sequence dysregulation being tied to disease

Given the many processes that have been outlined previously that are dependent on RNA functionality, it should come as no surprise that there exist genetic diseases and forms of cancer where disruption of transcript functionality is observed. Many of the more deletarious mutations alter the blueprints for protein production found in mRNA, whereas other mutations act through alternate means to disrupt gene regulation.

Many diseases that act through RNA dysregulation act to alter the the resultant protein product made from a transcript. Perhaps the most obvious example of RNA transcript disruption leading to disease is when an alteration of codon content in the coding sequence leads some change in the resultant protein that is translated. Multiple diseases are tied to the alteration of the coding region, which as mentioned before, is itself highly conserved sequence content. Within these coding regions have been found multiple examples of

sequence variation that are synonymous (do not change the sequence of the coding region), but have been reported to be functional disease associated mutations. Regions that are particularly sensitive to synonymous variants in the coding region are those that are involved in splicing. Study of natural synonymous human genetic variation has shown that there is a depletion of their overlap with exon splicing enhancer regions [163]. Plenty of examples exist of synonymous point mutations in exons, as well as point mutations inside intronic regions in pre-mRNA, that alter splicing. Such mutations have been found in disease-relevant genes in patients with colorectal cancer, Phenyketonuria, Dementia, Frazier Syndrome and multiple sclerosis [164, 165]. Disease states that are particularly well known to be associated with splicing defects are Duchene Muscular Dystrophy (caused by missplicing of the DMD transcript) and Spinal Muscular Atrophy (cause by the misplicing of the SMN1 gene during transcription, which leads to propagated aberrant splicing) [97]. Additionally, examples exist of mutations in small nuclear RNA that alter splicing. While examples of mutations in snRNAs are few and far between (likely due to their strong deletarious effect), induction of mutations in mouse U2 snRNA has been shown to lead to severe ataxia and neurogeneration [63]. Additionally, mutations in the minor spliceosome snRNA U4atac have been found in patients with microcephalic osteodysplastic primordial dwarfism type I (MOPD I) [62].

There are many additional examples of disease associated mutations that act through the alteration of different mechanisms of gene regulation. SnoRNA transcripts, which are typically involved in transcript modification as well as additional roles still being discovered, have had several diseases tied to their disregulation. The most well-known is Pradar-Willi syndrome (tied to the deletion of a cluster containing several snoRNAs) [166]. In miRNA, mutations can alter both transcript maturation and sequence preference in target binding. In changing target preference, mutations in miRNA are particularly deletarious when they are found in the seed region. Several known examples of disease-associated miRNA mutations exist, including the first disease-associated SNP found in an miRNA (which is associated with hearing loss) [167]. Disregulation of miRNA is strongly tied to various forms of cancer as well, with multiple miRNAs found to be differentially expressed in different cancers, and mutations that alter miRNA/target binding affinity, such as in the case of a SNP altering KRAS/let-7 binding [168–170]. The prospect of mutations in lncRNAs is particularly intriguing due to

their variety of functionalities, as well as the network level regulatory influence that a subset of these RNAs can exert. In spite of this, only a few functional mutations in lncRNAs have been found. A well-known example is the lncRNA TERC (RNA component of the telomere extending telomerase RNP), where mutations are found in individuals wtih Dyskeratosis Congenita, a premature aging disease [171]. SNPs within the gene for the lncRNA ANRIL that are associated with atherosclerotic vascular disease have additionally been found to be functional [172]. Finally, many ties have been made between lncRNA and cancer. A particular lncRNA, MALAT1, has drawn attention for being frequently mutated in colorectal and breast cancers [173].

1.9.2 Examples exist of disease-associated mutations in RNA predicted to change structure

While most work into the mechanisms through which these disease-associated mutations act has focused on looking for the disruption of conserved sequence content, there do exist examples of mutations where it was found that the mutations may have an effect on higher- order structure. Examples can be found in a RNA classes involved in protein production, as well as classes that are regulatory in nature.

There are several classic examples that are required discussion when covering rare mutations in patients that are though to act through the disruption of RNA structure. One of the classic examples is the repeat expansion found in patients with myotonic dystrophy. The repeat region, consisting normally of 5-38 CUG repeats in the the 3’UTR of DMPK mRNA, is expanded in myotonic dystrophy to above 50 repeats. Normally, the repeats form a stem region with multiple interior bulges, which has been shown to bind the RNA binding proteins CUGBP1 and MBNL1 [174]. When the repeat is expanded this structure motif grows in size, and thus recruits these proteins to the point where they are drawn away from subcellular locations where they presence is required, leading to pathogenesis of the disease [174]. In at least one patient, The Selenocystein elements in SEPN1 mRNA that were previously described have also been found to be mutated in particular patients with spinal muscular atrophy, and has been shown to abolish the binding of the protein SBP2 and this element that usually occurs [175]. Another well known example of a disease associated

with mutations in functional RNA structure is hyperferritinemia cataract syndrome. These elements, found in UTRs of various mRNAs whose protein products are involved in iron metabolism, are particularly sensitive to mutations that occur within these structured regions, as the proper formation of the IRE stemloop is important to the completion of its function (recruitment and binding of the Iron Regulatory Protein) [156, 176–178]. These mutations are analyzed in-depth in further chapters, and high resolution chemical mapping displaying the structure change that a subset of these mutations bring about is provided [124, 179].

There are also likely examples where functional RNA structure change occurs in spite of assumptions that the functional effect of mutations is through an alternate mechanism. A good example involves mutations in the PTEN gene found in individuals with Cowden Syndrome, characterized by tumor-like growths in epidermal regions and a significantly increased cancer susceptibility. A subset of these mutations are found in the 5’ end of the gene, where their functional effect was throught to be the alteration of promoter activity [180]. However, an analysis of 5 mutations detected in Cowden Syndrome patients within the promoter region in question showed that they did not fall into known transcription factor binding sites, and that PTEN mRNA levels remained constant in sequences with the mutations. However, it was shown that translation efficiency was significantly altered in 3 out of 5 mutations. RNA secondary structure prediction work done showed that a change in predicted MFE structure between wildtype and mutant sequences in the PTEN 5’UTR [181]. Examples of other mutations (often in the 5’UTR region) whose predicted functional mechanism is through the alteration of transcriptional activity, but have been predicted to significantly change RNA secondary structure, have been identified in our work described in further chapters [124].