Live Chat Support Software
800.227.0627

RNA nucleotide repeats, Poly(A) RNA, and phase separation

The expansion of short nucleotide repeats is the cause of several neurological and neuromuscular disorders. Disease-causing short nucleotide repeats occur in the coding or non-coding regions of RNA transcripts. However, in all of the nucleotide repeat expansion disorders identified to date, only a critical number of repeats appear to cause the disease. Expanded repeats in transcripts can cause cellular toxicity and neurodegeneration by altering the splicing machinery.

Presently it is thought that toxic RNAs interact with different RNA binding proteins to produce disease, known as the "trans-dominant" model of RNA toxicity. The model proposes that the interaction of mutant RNA with RNA binding proteins interferes with the functions of the interacting proteins, leading to abnormalities in the pathways regulated by the RNA binding proteins.


Since 1992, scientists know that a CTG repeat expansion in the 3’-untranslated region (3’-UTR) of a protein kinase gene causes myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy. Southern blot, polymerase chain reaction (PCR)-based methods, and direct sequencing of PCR amplified CTG repeats allow the detection of these repeats in patients diagnosed with congenital myotonic dystrophy. 

The expansion of a hexanucleotide microsatellite DNA repeat (GGGGCC) in an intron of the C90RF72 gene is the cause of two neurodegenerative diseases. These are frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Unfortunately, presently there is no cure for these two diseases.

Jain & Vale in 2017 showed that repeat expansions create templates for multivalent base-pairing. In purified RNA of this type, a sol-gel transition occurs at a similar critical repeat number as that found in diseases. According to the researchers, in cells, RNA foci form by phase separation of the repeat-containing RNA. However, agents that disrupt RNA gelation in vitro can dissolve repeat-containing RNAs. Jain & Vale's suggested that sequence-specific gelation of RNAs may contribute to a specific neurological disease like in protein aggregation disorders.

Earlier studies by Eisenberg & Felsenfeld found that polyriboadenylic acid (poly (A) RNA) undergoes reversible phase separation at neutral pH. Poly (A) RNA is soluble at low and high temperatures but only partially miscible in a range of 35 to 40 °C. However, the range extent varies with molecular weight and polymer and salt concentrations. Poly(A) RNA precipitate at 35 to 40 °C but will remain soluble outside of that range. At a high temperature, the poly(A) polymer appears as a compact molecule. At a low temperature as an extended molecule.


This phenomenon's driving force appears to be base stacking, in which nucleic acid bases stack with their planes parallel to one another. However, Eisenberg & Felsenfeld's observation suggested structure formed by a non-co-operative process. 
As a result, poly(A) molecules of different lengths phase-separated at different conditions allowing the fractionation of poly(A) mixtures into homogenous solutions.

More recently, phase separation is emerging as a ubiquitous process for the compartmentalization and concentration of biomolecules at specific cell locations. Many many proteins crucial for cell growth and development undergo phase separation. However, the observation that RNA too can experience phase separation is a recent concept in RNA biochemistry. Apparently, RNA can also instruct its distribution in cells independently of other cellular components.

Poly (A) can also be used as a carrier molecule for the purification of DNA and RNA from a variety of samples.

In 2018 Treeck et al. suggested that RNA self-assembly also contributes to the formation of stress granules. 

What are stress granules?

Stress granules form during the inhibition of translation initiation. Stress granules are higher-order assemblies of non-translating mRNAs. These are ubiquitous, non-membrane-bound assemblies of protein and RNA forming during the inhibition of translation initiation. Stress granules appear to play a role in stress response and gene regulation. Related ribonucleoprotein (RNP) granules exist in neurons and can affect synaptic plasticity. Mutations in RNA binding proteins or stress granule-remodeling complexes leading to an increased formation of stress granules appear to cause amyotrophic lateral sclerosis (ALS) and other degenerative disorders. The formation of stress granules can influence both tumor progression and viral infection.

Reference

DeJesus-Hernandez M, et al.; Expanded GGGGCC hexanucleotide repeat in non-coding region of C9ORF72 causes chromosome 9p-linked frontotemporal dementia and amyotrophic lateral sclerosis. Neuron. 2011;72(2):245–256. [PMC]

Jain A, Vale RD. RNA phase transitions in repeat expansion disorders. Nature. 2017 Jun 8;546(7657):243-247. doi: 10.1038/nature22386. Epub 2017 May 31. PMID: 28562589. [PMC]

Ohya K, Tachi N, Sato T, Kon S, Kikuchi K, Chiba S. Detection of the CTG repeat expansion in congenital myotonic dystrophy. Jpn J Hum Genet. 1997 Mar;42(1):169-80. [PubMed]

Orr HT. Toxic RNA as a driver of disease in a common form of ALS and dementia. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7533-4. [PMC]

Renton AE, Majounie E, Waite A, Simón-Sánchez J, Rollinson S, Gibbs JR, Schymick JC, Laaksovirta H, van Swieten JC, Myllykangas L, Kalimo H, Paetau A, Abramzon Y, Remes AM, Kaganovich A, Scholz SW, Duckworth J, Ding J, Harmer DW, Hernandez DG, Johnson JO, Mok K, Ryten M, Trabzuni D, Guerreiro RJ, Orrell RW, Neal J, Murray A, Pearson J, Jansen IE, Sondervan D, Seelaar H, Blake D, Young K, Halliwell N, Callister JB, Toulson G, Richardson A, Gerhard A, Snowden J, Mann D, Neary D, Nalls MA, Peuralinna T, Jansson L, Isoviita VM, Kaivorinne AL, Hölttä-Vuori M, Ikonen E, Sulkava R, Benatar M, Wuu J, Chiò A, Restagno G, Borghero G, Sabatelli M; ITALSGEN Consortium, Heckerman D, Rogaeva E, Zinman L, Rothstein JD, Sendtner M, Drepper C, Eichler EE, Alkan C, Abdullaev Z, Pack SD, Dutra A, Pak E, Hardy J, Singleton A, Williams NM, Heutink P, Pickering-Brown S, Morris HR, Tienari PJ, Traynor BJ. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron. 2011 Oct 20;72(2):257-68. [PMC]

Trcek, T. The curious behaviour of poly(A) RNA. Nat Rev Mol Cell Biol (2021). [Nature reviews]


Van Damme P, Robberecht W, Van Den Bosch L. Modelling amyotrophic lateral sclerosis: progress and possibilities. Dis Model Mech. 2017 May 1;10(5):537-549. [PMC]

Van Treeck, B. et al.; RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome. Proc. Natl Acad. Sci. USA 115, 2734–2739 (2018). [PNAS]

 

---...---