Mini-Review: Ribosome Profiling
Introduction
TDP-43 is by far the most common factor associated with ALS, with aggregations of the protein being observed in in ~97% of cases, be they sporadic or familial in origin (2). Recent evidence suggests that the impact of these aggregates results from a loss of normal function of this protein, as opposed to a toxic gain-of-function of the aggregates themselves (5). Yet, somewhat frustratingly, TDP-43 has been shown to have a variety of functions, ranging from transcriptional repression (6) to exon splicing (7), as well as mRNA transportation (8), thus making clear conclusions to the impact of a loss-of-function difficult. However, with particular reference to translation in neurons, TDP-43 has recently been found to bind to and transport ribosomal protein mRNAs to the axons of neurons, where they are then locally translated and incorporated into local, axon-based and/or synaptic bouton-based ribosomes. In human sporadic ALS samples, these mRNAs were indeed found to be reduced in expression in pyramidal tract of the medulla oblongata, where axons of motor neurons are located (8). Adding to its role in translation, it has been demonstrated that, under conditions of stress, this protein was found to associate with stalled ribosomes, and that this interaction likely contributes to cell survival (9).
Delving further in this vein, a global proteomic study found that TDP-43-interacting proteins largely cluster into two distinct groups, a smaller nuclear-based splicing cluster and a comparatively larger cytosolic-based translational cluster (10). TDP-43 alters ribosome association of certain transcripts (11), and further ribosome profiling experimentation has determined that TDP-43 translationally upregulates the translation of specific transcripts, such as Camta1, Mig12, and Dennd4a (12). However, other studies have demonstrated downregulated translation, at the level of individual transcripts (13), but also globally (14). One interesting point to note is that inhibitors of eIF2α phosphorylation appears to rescue TDP-43 toxicity (15). EIF2α phosphorylation is known to downregulate global translation, implying that an increase in translational functionality may be important in rescuing neurons in these conditions. In any case, it is clear that TDP-43 has a wide range of interactors, both at the protein and RNA levels, thus mystifying its more detailed role. Insights will likely be gained from a wider analysis of these interactors, with the hope that their individual functions will provide clues to the more granular role TDP-43 may play, under a host of different circumstances. However, that it has a significant role in the translational landscape of ALS is clear.
C9orf72
C9orf72 refers to its location at open reading frame 72 on chromosome 9. In ALS, this is the site of a massive intronic GGGGCC hexanucleotide repeat expansion, and this is the most common mutation associated with familial ALS (16). In most healthy individuals, this repeat typically occurs only twice, however, in those that go on to display the phenotype of the disease, this repeat may occur up to hundreds or even thousands of times (17), and is typically associated with a gain-of-toxicity (18). One of the impacts of these extensive repeats is the production of a number of dipeptide repeat proteins, initiated via repeat associated non-AUG translation. Multiple papers have associated these peptides with the induction of TDP-43 pathology (19-21), and thus C9orf72 mutations likely lead to some of the impacts attributed to TDP-43, described above. However, these extended repeats have been associated with other translational defects independent of TDP-43 pathology. Indeed, poly-PR and poly-GR dipeptides have been shown to form insoluble complexes with mRNA, limiting access of translational factors, leading to translation arrest and an overall reduction in global translation (22).
Numerous studies have demonstrated that these dipeptide repeat proteins bind to the ribosome, limiting their translational efficacy and inducing translational arrest (23-25), as well as impairing stress granule dynamics and reducing overall ribosome numbers. Importantly, overexpression of eIF1A was seen to rescue dipeptide-induced neural toxicity, strongly implicating that translation is majorly impacted by such dipeptide repeats (25). Furthermore, the structural basis for these mechanisms has been partially elucidated, with both poly-PR and poly-GR found to bind to the polypeptide exit tunnel of the ribosome, extending into the peptidyl-transferase center, thus inhibiting peptidyl-transferase activity (26). Additionally, research has indicated there may be no evolved regulatory mechanism for resolving such stalling (27), which may explain their high toxicity at nano-molar concentrations (26).
FUS
Fused in sarcoma, or FUS, is an RNA-binding protein. Its name originates from its initial discovery, where the 5’ end of this gene undergoes chromosomal translocation, “fusing” with another gene, CHOP, in human cancers. However, in 2009, a study implicated mutations in this gene with a type-6 ALS phenotype (28). It is currently unclear whether mutant FUS leads to a loss of normal function, or a toxic gain of function. FUS knockout mice do go on to display phenotypes of neurodegenerative conditions, although these appear to be distinct from ALS (29). As such, it is likely that these mutated proteins are detrimental in of themselves. Mutant FUS mislocalised from the nucleus to the cytoplasm, where it forms cytoplasmic inclusions/liquid-liquid phase separation (30). With regard to translation, luciferase and methionine incorporation assays have demonstrated that mutant FUS supressed mRNA translation, with FUS cytoplasmic inclusions also being shown to be associated with stalled ribosomes (31). A second study identified the cytoplasmic inclusions contained FMRP-bound RNAs which are then translationally repressed (32). Such activity has also been linked to a hyperactivation of the process of nonsense-mediated decay, leading to significant mRNA degradation (31). Furthermore, non-mutated FUS has been found to supress translation through mTOR-dependant signalling, and mutated FUS may possibly contribute to pathological translation suppression through its mislocalisation from the nucleus to the cytoplasm (33).
Others
Angiogenin
Angiogenin is a protein most well-known for its stimulation of blood vessel formation. Perhaps not surprisingly, with its angiogenic properties, it has also been associated with the development of cancer. However, it has also been associated with the development of ALS (34). Multiple studies have associated the function of this gene with the stimulation of rRNA transcription (35, 36), which makes sense in the context of the necessary cell proliferation required for angiogenesis. Furthermore, mutations in this gene have been linked to a loss-of-function regarding its role in ALS, rather than a toxic gain-of-function (37, 38), implicating reduced rRNA transcription in angiogenin-associated ALS, and quite possibly reduced ribosome biogenesis.
Ataxin-2
Ataxin-2 is a ubiquitously expressed protein, with many purported physiological and pathophysiological functions, ranging from embryonic development, cell proliferation, fertility, and obesity (39). Many of these functions are intrinsically fuelled by protein synthesis, which is of course fundamentally underpinned by translation. Ataxin-2 is perhaps most well-known for its association with spinocerebellar ataxia type 2, wherein the gene contains a significant amount of abnormal CAG repeat expansions. However, more recently, such repeats in this gene have also been associated with ALS, with both disorders sharing similar symptomology (40, 41). This gene also has significant associations with TDP-43, with both proteins forming a complex dependant on RNA binding (40). ALS-associated ataxin-2 repeats have been shown to upregulate TDP-43 pathology (42), and therapeutic approaches downregulating ataxin-2 markedly reduce TDP-43 pathology as well as dramatically extending survival times in a mouse model of ALS (43).
p97
P97, otherwise known as VCP, is a protein typically associated with the ubiquitination process. In 2010, it was linked to approximately 1-2% of familial ALS cases (44), and its toxicity was hypothesised to be partially mediated through TDP-43, with which it is an interactor. However, it also has specific functions relating to translational events, such as the promotion of cellular internal ribosome entry site (IRES) translation (45) and degradation of ribosome-bound aberrant nascent polypeptides (46). Knockdown of p97 has also been demonstrated to lead to a reduction in global translation (47), and thus any loss-of-function mutations in this gene may contribute to translational abnormalities.
Conclusions
ALS is a complex disease, with a plethora of associated genes, many of which are not mentioned in this piece. Additionally, even within specific genes, alternative mutations can lead to significantly different phenotypes (48). As such, ALS constitutes an umbrella term, describing broad, yet similar, characteristic symptoms. With such a wide range of associated genes, there is likely no one singular mechanism of action. Indeed, it must be noted in this piece, one of the most significant genes associated with ALS, SOD1 (linked to 12% of fALS and 1-2% sALS) (49), has comparatively little translatomic association. It also has little to no association with pathological TDP-43 (50), the molecule associated with the vast majority of ALS cases (2). Thus, to attribute any one mechanism to this disease would be unwise. However, the translational links possessed by a great many of the genes most associated with this disease are clear, and likely represent a viable mechanism for a significant portion of cases. As such, research into therapeutics to rescue the translational impairments observed in models of ALS may constitute an exciting and rewarding area of investigation, and should be explored further.
References
1. Talbott EO, Malek AM, Lacomis D. The epidemiology of amyotrophic lateral sclerosis. Handb Clin Neurol. 2016;138:225-38.
2. Tan RH, Ke YD, Ittner LM, Halliday GM. ALS/FTLD: experimental models and reality. Acta Neuropathol. 2017;133(2):177-96.
3. Cestra G, Rossi S, Di Salvio M, Cozzolino M. Control of mRNA Translation in ALS Proteinopathy. Front Mol Neurosci. 2017;10:85.
4. Lehmkuhl EM, Zarnescu DC. Lost in Translation: Evidence for Protein Synthesis Deficits in ALS/FTD and Related Neurodegenerative Diseases. Adv Neurobiol. 2018;20:283-301.
5. Vanden Broeck L, Callaerts P, Dermaut B. TDP-43-mediated neurodegeneration: towards a loss-of-function hypothesis? Trends in Molecular Medicine. 2014;20(2):66-71.
6. Ou SH, Wu F, Harrich D, García-Martínez LF, Gaynor RB. Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. J Virol. 1995;69(6):3584-96.
7. Buratti E, Brindisi A, Pagani F, Baralle FE. Nuclear factor TDP-43 binds to the polymorphic TG repeats in CFTR intron 8 and causes skipping of exon 9: a functional link with disease penetrance. Am J Hum Genet. 742004. p. 1322-5.
8. Nagano S, Jinno J, Abdelhamid RF, Jin Y, Shibata M, Watanabe S, et al. TDP-43 transports ribosomal protein mRNA to regulate axonal local translation in neuronal axons. Acta Neuropathol. 2020;140(5):695-713.
9. Higashi S, Kabuta T, Nagai Y, Tsuchiya Y, Akiyama H, Wada K. TDP-43 associates with stalled ribosomes and contributes to cell survival during cellular stress. J Neurochem. 2013;126(2):288-300.
10. Freibaum BD, Chitta RK, High AA, Taylor JP. Global Analysis of TDP-43 Interacting Proteins Reveals Strong Association with RNA Splicing and Translation Machinery. Journal of Proteome Research. 2010;9(2):1104-20.
11. Lehmkuhl EM, Loganathan S, Alsop E, Blythe AD, Kovalik T, Mortimore NP, et al. TDP-43 proteinopathy alters the ribosome association of multiple mRNAs including the glypican Dally-like protein (Dlp)/GPC6. Acta Neuropathologica Communications. 2021;9(1):52.
12. Neelagandan N, Gonnella G, Dang S, Janiesch PC, Miller KK, Küchler K, et al. TDP-43 enhances translation of specific mRNAs linked to neurodegenerative disease. Nucleic Acids Research. 2019;47(1):341-61.
13. Majumder P, Chen YT, Bose JK, Wu CC, Cheng WC, Cheng SJ, et al. TDP-43 regulates the mammalian spinogenesis through translational repression of Rac1. Acta Neuropathol. 2012;124(2):231-45.
14. Russo A, Scardigli R, La Regina F, Murray ME, Romano N, Dickson DW, et al. Increased cytoplasmic TDP-43 reduces global protein synthesis by interacting with RACK1 on polyribosomes. Human Molecular Genetics. 2017;26(8):1407-18.
15. Kim HJ, Raphael AR, LaDow ES, McGurk L, Weber RA, Trojanowski JQ, et al. Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models. Nat Genet. 2014;46(2):152-60.
16. Smith BN, Newhouse S, Shatunov A, Vance C, Topp S, Johnson L, et al. The C9ORF72 expansion mutation is a common cause of ALS+/−FTD in Europe and has a single founder. European Journal of Human Genetics. 2013;21(1):102-8.
17. Iacoangeli A, Al Khleifat A, Jones AR, Sproviero W, Shatunov A, Opie-Martin S, et al. C9orf72 intermediate expansions of 24–30 repeats are associated with ALS. Acta Neuropathologica Communications. 2019;7(1):115.
18. Jiang J, Zhu Q, Gendron TF, Saberi S, McAlonis-Downes M, Seelman A, et al. Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs. Neuron. 2016;90(3):535-50.
19. Nonaka T, Masuda-Suzukake M, Hosokawa M, Shimozawa A, Hirai S, Okado H, et al. C9ORF72 dipeptide repeat poly-GA inclusions promote intracellular aggregation of phosphorylated TDP-43. Hum Mol Genet. 2018;27(15):2658-70.
20. Cook CN, Wu Y, Odeh HM, Gendron TF, Jansen-West K, Del Rosso G, et al. C9orf72 poly(GR) aggregation induces TDP-43 proteinopathy. Sci Transl Med. 2020;12(559).
21. Ryan S, Rollinson S, Hobbs E, Pickering-Brown S. C9orf72 dipeptides disrupt the nucleocytoplasmic transport machinery and cause TDP-43 mislocalisation to the cytoplasm. Scientific Reports. 2022;12(1):4799.
22. Kanekura K, Yagi T, Cammack AJ, Mahadevan J, Kuroda M, Harms MB, et al. Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation. Hum Mol Genet. 2016;25(9):1803-13.
23. Zhang Y-J, Gendron TF, Ebbert MTW, O’Raw AD, Yue M, Jansen-West K, et al. Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nature Medicine. 2018;24(8):1136-42.
24. Hartmann H, Hornburg D, Czuppa M, Bader J, Michaelsen M, Farny D, et al. Proteomics and C9orf72 neuropathology identify ribosomes as poly-GR/PR interactors driving toxicity. Life Sci Alliance. 2018;1(2):e201800070.
25. Moens TG, Niccoli T, Wilson KM, Atilano ML, Birsa N, Gittings LM, et al. C9orf72 arginine-rich dipeptide proteins interact with ribosomal proteins in vivo to induce a toxic translational arrest that is rescued by eIF1A. Acta Neuropathol. 2019;137(3):487-500.
26. Loveland AB, Svidritskiy E, Susorov D, Lee S, Park A, Zvornicanin S, et al. Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM. Nature Communications. 2022;13(1):2776.
27. Kriachkov V, McWilliam HEG, Mintern JD, Amarasinghe SL, Ritchie M, Furic L, et al. Arginine-rich C9ORF72 ALS Proteins Stall Ribosomes in a Manner Distinct From a Canonical Ribosome-Associated Quality Control Substrate. bioRxiv. 2022:2022.02.09.479805.
28. Vance C, Rogelj B, Hortobágyi T, De Vos KJ, Nishimura AL, Sreedharan J, et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009;323(5918):1208-11.
29. Kino Y, Washizu C, Kurosawa M, Yamada M, Miyazaki H, Akagi T, et al. FUS/TLS deficiency causes behavioral and pathological abnormalities distinct from amyotrophic lateral sclerosis. Acta Neuropathol Commun. 2015;3:24.
30. Yasuda K, Clatterbuck-Soper SF, Jackrel ME, Shorter J, Mili S. FUS inclusions disrupt RNA localization by sequestering kinesin-1 and inhibiting microtubule detyrosination. J Cell Biol. 2017;216(4):1015-34.
31. Kamelgarn M, Chen J, Kuang L, Jin H, Kasarskis EJ, Zhu H. ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay. Proceedings of the National Academy of Sciences. 2018;115(51):E11904.
32. Birsa N, Ule AM, Garone MG, Tsang B, Mattedi F, Chong PA, et al. FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation. Sci Adv. 2021;7(30).
33. Sévigny M, Bourdeau Julien I, Venkatasubramani JP, Hui JB, Dutchak PA, Sephton CF. FUS contributes to mTOR-dependent inhibition of translation. J Biol Chem. 2020;295(52):18459-73.
34. Greenway MJ, Alexander MD, Ennis S, Traynor BJ, Corr B, Frost E, et al. A novel candidate region for ALS on chromosome 14q11.2. Neurology. 2004;63(10):1936.
35. Sheng J, Yu W, Gao X, Xu Z, Hu GF. Angiogenin stimulates ribosomal RNA transcription by epigenetic activation of the ribosomal DNA promoter. J Cell Physiol. 2014;229(4):521-9.
36. Tsuji T, Sun Y, Kishimoto K, Olson KA, Liu S, Hirukawa S, et al. Angiogenin Is Translocated to the Nucleus of HeLa Cells and Is Involved in Ribosomal RNA Transcription and Cell Proliferation. Cancer Research. 2005;65(4):1352-60.
37. Wu D, Yu W, Kishikawa H, Folkerth RD, Iafrate AJ, Shen Y, et al. Angiogenin loss-of-function mutations in amyotrophic lateral sclerosis. Annals of Neurology. 2007;62(6):609-17.
38. Padhi AK, Jayaram B, Gomes J. Prediction of Functional Loss of Human Angiogenin Mutants Associated with ALS by Molecular Dynamics Simulations. Scientific Reports. 2013;3(1):1225.
39. Ostrowski LA, Hall AC, Mekhail K. Ataxin-2: From RNA Control to Human Health and Disease. Genes (Basel). 2017;8(6).
40. Elden AC, Kim HJ, Hart MP, Chen-Plotkin AS, Johnson BS, Fang X, et al. Ataxin-2 intermediate-length polyglutamine expansions are associated with increased risk for ALS. Nature. 2010;466(7310):1069-75.
41. Sproviero W, Shatunov A, Stahl D, Shoai M, van Rheenen W, Jones AR, et al. ATXN2 trinucleotide repeat length correlates with risk of ALS. Neurobiol Aging. 2017;51:178.e1-.e9.
42. Hart MP, Gitler AD. ALS-associated ataxin 2 polyQ expansions enhance stress-induced caspase 3 activation and increase TDP-43 pathological modifications. J Neurosci. 2012;32(27):9133-42.
43. Becker LA, Huang B, Bieri G, Ma R, Knowles DA, Jafar-Nejad P, et al. Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice. Nature. 2017;544(7650):367-71.
44. Johnson JO, Mandrioli J, Benatar M, Abramzon Y, Van Deerlin VM, Trojanowski JQ, et al. Exome sequencing reveals VCP mutations as a cause of familial ALS. Neuron. 2010;68(5):857-64.
45. Hundsdoerfer P, Thoma C, Hentze MW. Eukaryotic translation initiation factor 4GI and p97 promote cellular internal ribosome entry sequence-driven translation. Proceedings of the National Academy of Sciences. 2005;102(38):13421-6.
46. Verma R, Oania RS, Kolawa NJ, Deshaies RJ. Cdc48/p97 promotes degradation of aberrant nascent polypeptides bound to the ribosome. eLife. 2013;2:e00308.
47. Lee SH, McCormick F. p97/DAP5 is a ribosome-associated factor that facilitates protein synthesis and cell proliferation by modulating the synthesis of cell cycle proteins. The EMBO Journal. 2006;25(17):4008-19.
48. Berdyński M, Miszta P, Safranow K, Andersen PM, Morita M, Filipek S, et al. SOD1 mutations associated with amyotrophic lateral sclerosis analysis of variant severity. Scientific Reports. 2022;12(1):103.
49. Renton AE, Chiò A, Traynor BJ. State of play in amyotrophic lateral sclerosis genetics. Nat Neurosci. 2014;17(1):17-23.
50. Mackenzie IR, Bigio EH, Ince PG, Geser F, Neumann M, Cairns NJ, et al. Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann Neurol. 2007;61(5):427-34.