Structural insight of r(CGG) motif and small molecule based therapeutics development for the expanded CGG repeats RNA associated neurological disorders

Abstract

Ribonucleic acid (RNA) is a unique biological polymer for cellular regulation and maintenance. A central principle about RNA is that it transfers the biological information from DNA via RNA to protein. Since many years, before the discovery of its catalytic activity, RNA is only known for its three primary functions such as synthesis xerox of the DNA (mRNA), coupler between the amino acid (protein building block) & genetic codes and ribosomal RNA. Such a new discovery of RNA, its catalytic activity has opened the new universe for the scientific community to study the shaded cellular mechanisms. For the effective function of the cell, rigorous and controlled expression of protein-coding (mRNA) and noncoding RNAs is imperative. In addition, different types of RNAs have been discovered which have a crucial role in cell regulatory processes. RNA has been categorized based on their structural as well as functional roles in cellular events. For example, transfer RNA (t-RNAs), ribosomal RNA (r-RNAs), 7SL/SRP RNA and microRNAs (miRNAs) involved in translation processes. Similarly, other RNAs like small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) involved being participating in RNA processing. Some different sets of RNA molecules involved in other cellular processes such as telomere-associated RNAs, Ribonuclease P RNase, and mitochondrial RNA processing (MRP) RNase. Apart from all these, few RNAs are very less elaborated with unknown functions such as vault RNAs, Y RNAs, Piwi-interacting RNAs (piRNAs). Typically, RNAs possess different topological structure to deliver specific functions. Topological structure with precise folding of RNAs could be a deciding factor for the phenotypic outcomes. Riboswitches, microRNAs and siRNAs are the epitome for the importance of RNA secondary structure in which canonical and non-canonical base paring places a central role in small RNA processing that leads to the recognition and successive regulation of selected messenger RNA. Thus, the formation of secondary structures of RNA is crucial to regulate cellular processes. However, changes in the base-pairing are simple but significant for not only for the regulatory RNA (microRNAs, siRNAs) but also for mRNA and long noncoding RNAs. Unlikely, unusual secondary hairpin structures formation by mRNAs is associated with various human diseases. These uncommon hairpin structures are formed due to the unstable and dynamic expansion of repetitive sequences in the human genome. These repetitive sequences are simple sequence repeats (SSR), or tandem repeats also known as microsatellites, and almost 30 percent of the human genome constituted these microsatellites. Alternation in the sequence and the length of repeatome gives rise to diversity in the species during an evolutionary period. Although, a variation of these simple repetitive DNA sequences beyond threshold limit results human disorders. These repetitive sequences are present in the coding (exons) and noncoding (5'UTR, introns, and 3'UTR) region of the gene and could be tri, tetra, penta, hexa and deca repeats in nature. Trinucleotide repeat (TNR) disorders occur when the number of triplet repeats is higher in mutated genes compared to a number of triplets found in the normal gene. Additionally, TNRs are genetically inherited in nature and become severe with the successive generation. Increase in number of TNRs beyond the specific limit completely changes the expression and functional profile of the gene. The length of the triplet repeats shows a very striking genotypic-phenotypic correlation with disease pathology. Longer the repeat length leads to worsening the disease condition and early onset of symptoms. Depending on the type of trinucleotide expansion and the location (coding and noncoding) on the gene caused different neurological disorders. CGG nucleotide expansion at 5′UTR of FMR1 gene causes Fragile X syndrome (FXS), Fragile X-associated tremor/ataxia syndrome (FXTAS) and Fragile X-associated primary ovarian insufficiency (FXPOI). Expansion of GCC repeats at 5′UTR of FMR2 gene cause Fragile XE mental retardation syndrome (FRAXE). Similarly, expansion of CAG repeats at coding, and noncoding region of the different genes causes different neurodegenerative diseases including, Spinocerebellar ataxia type (1, 2, 3, 6, 7, 12 and 17), Huntington disease, Huntington disease-like 2, Spinobulbar muscular atrophy and Dentatorubral-pallidoluysian atrophy. Further, CTG and CCTG repeat expansion causes Myotonic dystrophy type 1 (DM1), Spinocerebellar ataxia 8 and Myotonic dystrophy type 2 (DM2) respectively. Likewise, GAA repeat causes Friedreich’s ataxia. All neurological disorders show a different pattern of TNRs expansion and the proposed models for the repeat expansion suggested that formation of intermediate looped structure incorporate in the DNA. However, why some repeats expansion occurs more in the genome than other repeats are still unanswered. The previous report demonstrated that DNA repair and replication mediates the repeat expansion through polymerase slippage mechanism. All these neurological disorders possess common pathogenic mechanisms 1) RNA gain of function in which mRNA transcript form stable secondary structure that sequester several RNA binding protein for e.g. DiGeorge syndrome critical region 8 (DGCR8), DROSHA, Src associated in mitosis of 68 kDa (Sam68), Heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2/B1), CUGBP1 and Pur. Thus, sequestration of these protein results in the formation of nuclear RNA foci and also influence splicing regulation. Another pathogenic mechanism includes protein gain of function in which repeat expansion promotes the formation of homopolymeric protein aggregates e.g. polyglutamine (polyQ) and polyglycine (polyG). Additionally, protein loss of function mechanism occurs due to over-expansion of TNRs on the corresponding gene induce epigenetic silencings such as Frataxin and FMRP. FXTAS is caused by the CGG trinucleotide expansion on the 5′UTR of fragile X mental retardation 1 (FMR1) gene on the long arm of the X chromosome at Xq27.3, code for fragile X mental retardation protein (FMRP). It is a late-onset monogenic neurological disorder which affects adult over the age of 50. The numbers of CGG repeats vary 5-54 times at 5′UTR of FMR1 gene in fragile X carrier. However, due to instability of CGG repeats across generations lead to enhancement in the repeat expansion to 55-200, called fragile X premutation (PM) carriers. The prevalence rate of FXTAS is approximately 1 in 400-800 males and 1 in 260 females. The probability of developing FXTAS in older age (50) is 40% of males and 8-16% of females. Male are more prone for FXTAS than female as extra X chromosome compensate the disease condition. Initially, premutation carriers were thought to be unaffected. However, it is now known to be associated with a huge spectrum of neurodegenerative and clinical symptoms. The major clinical symptoms include with premutation carrier is intention tremor and gait ataxia. Minor clinical symptoms include memory loss, dementia, cognitive decline, Parkinsonism, and psychiatric problems. Neurohistopathological hallmark of FXTAS includes the formation of ubiquitin-positive inclusion bodies inside the brain cells (neurons and astrocytes) of animal and post-mortem brain tissues. Immunohistochemistry and mass spectrometric analysis of these inclusion bodies disclosed the presence of more than 30 inclusions associated protein for example- DGCR8, DROSHA, Sam68, hnRNPA2/B1, CUGBP1, Pur, neurofilament, and lamina A/C. Similar intracellular inclusion bodies are also found in the non-CNS organs of the patient, including, thyroid, kidney, heart, pituitary and adrenal gland. Further, degeneration was also reported in the cerebellum, including Purkinje fibers, nerve cells, glial cells, and spongiosis of cerebellar white matter. Mitochondrial dysfunction, disruption of nuclear lamina structure, loss of dendrite and dendritic spine morphology are other pathological features, examined in FXTAS.