RNA and disease

Abstract

Cellular functions depend on numerous protein-coding and noncoding RNAs and the RNA-binding proteins associated with them, which form ribonucleoprotein complexes (RNPs). Mutations that disrupt either the RNA or protein components of RNPs or the factors required for their assembly can be deleterious. Alternative splicing provides cells with an exquisite capacity to fine-tune their transcriptome and proteome in response to cues. Splicing depends on a complex code, numerous RNA-binding proteins, and an enormously intricate network of interactions among them, increasing the opportunity for exposure to mutations and misregulation that cause disease. The discovery of disease-causing mutations in RNAs is yielding a wealth of new therapeutic targets, and the growing understanding of RNA biology and chemistry is providing new RNA-based tools for developing therapeutics.

Figure 1. The mRNA biogenesis pathway and post-transcriptional gene regulation

Overview of the dynamic interactions of the major RNPs involved in pre-mRNA processing, mRNA biogenesis and regulation of gene expression. The nascent transcript (pre-mRNA), capped with a 7-methyl guanosine (m⁷G) cap, is bound by hnRNP proteins and SR (serine-arginine-rich domain-containing) proteins. Small nuclear ribonucleoprotein particles (snRNPs) bind to the splice sites at the 5′ and 3′ ends of introns. The spliced introns are excised as lariats, which are then debranched and degraded. The exon junction complex (EJC) assembles on spliced mRNA ~20 nucleotides upstream of joined exons, followed by the export of the mRNA to the cytoplasm. MicroRNAs (miRNAs) in the form of ribonucleoprotein complexes (miRNPs) bind to target mRNAs to regulate translation and mRNA stability. The poly(A)-binding protein (PABP) binds to the poly(A) tail of cytoplasmic mRNAs. The SR domains are indicated with a star. cEJC indicates the remaining stable EJC on mRNA in the cytoplasm, which is removed by translating ribosomes.

Figure 2. Mutations disrupting the cis-acting splicing code cause splicing defects and disease

A. The motor neuron degenerative disease SMA results from deletion of the SMN1 gene. The remaining SMN2 gene has a C->T substitution in exon 7 that inactivates an exonic splicing enhancer (ESE), and creates an exonic splicing silencer (ESS), leading to exon 7 skipping and a truncated protein (SMNΔ7). B. A T->A substitution in exon 31 of the dystrophin gene simultaneously creates a premature termination codon (STOP) and an ESS, leading to exon 31 skipping. This mutation causes a mild form of DMD because the mRNA lacking exon 31 produces a partially functional protein. C. Mutations within and downstream of exon 10 of the MAPT gene encoding the tau protein affect splicing regulatory elements and disrupt the normal 1:1 ratio of mRNAs including or excluding exon 10. This results in a perturbed balance between tau proteins containing either four or three microtubule-binding domains (4R-tau and 3R-tau, respectively), causing the neuropathological disorder FTDP-17. The example shown is the N279K mutation which enhances an ESE function promoting exon 10 inclusion and shifting the balance toward increased 4R-tau. D. Polymorphic (UG)m(U)n tracts within the 3′ splice site of the CFTR gene exon 9 influence the extent of exon 9 inclusion and the level of full-length functional protein, modifying the severity of cystic fibrosis (CF) caused by a mutation elsewhere in the CFTR gene.

Figure 3. RNA gain-of-function

A. Microsatellite expansions within transcribed regions with confirmed or potential RNA gain-of-function effects. The type and number of nucleotide repeats are indicated. (Fragile X-associated tremor ataxia syndrome, FXTAS; premature ovarian insufficiency, POI; Huntington’s disease, HD; spinocerebellar ataxias, SCAs; dentatorubral pallidoluysian atrophy, DRPLA; spinal and bulbar muscular atrophy, SBMA; Huntington’s disease like 2, HDL2). B. Sequestration of MBLN1 in myotonic dystrophy (DM1). C. Activation of protein kinase C (PKC) by the expanded CUG RNA induces hyperphosphorylation of CUGBP1, which stabilizes the protein.

Figure 4. Therapeutic approaches that use or target RNAs

A. Antisense oligonucleotides (AOs) or bifunctional AOs that have sequences complementary to exon 7 conjugated to splicing-enhancing effectors---e.g., serine-arginine (SR) peptide or an ESE that recruits SR proteins---are used to promote exon 7 inclusion of the SMN2 gene, decreasing the production of a truncated SMN protein (SMNΔ7) and increasing that of a full-length functional protein (SMN). B. Modified U1 or U7 snRNA-based vectors can carry complementary sequences to the targeting RNAs to achieve the desired splicing pattern. As shown here, genomic deletions (e.g., from exon 48 to 50) in dystrophin genes of DMD patients lead to a premature termination codon (STOP) in exon 51 caused by a frameshift. Antisense U7 snRNA targeting the 3′ splice site of exon 51 prevents the inclusion of this exon and restores the reading frame, and as a result, produces a partially functional protein. C. RNAi approaches are used to eliminate pathogenic mRNAs. siRNAs delivered to a mouse model of spinocerebellar ataxia type I (SCA1) elicit degradation and efficient knockdown of the disease-causing ataxin-1 mRNA that contains the expanded CAG repeats encoding polyglutamine (polyQ).