Relax, Don’T RAN Translate It
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Neuron Previews Relax, Don’t RAN Translate It Katherine M. Wilson,1,2 Bhavana Muralidharan,1,2,3 and Adrian M. Isaacs1,2,* 1UK Dementia Research Institute at UCL, Gower Street, London WC1E 6BT, UK 2Department of Neurodegenerative Disease, Queen Square UCL Institute of Neurology, London WC1N 3BG, UK 3Brain Development and Disease Mechanisms, Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, India, 560065 *Correspondence: [email protected] https://doi.org/10.1016/j.neuron.2019.11.014 The (GGGGCC)n repeat expansion in C9orf72, which is the most common cause of frontotemporal dementia and amyotrophic lateral sclerosis, is translated through repeat-associated non-AUG (RAN) translation. In this issue of Neuron, Cheng et al. (2019) report that the helicase DDX3X, which unwinds (or relaxes) RNA, sup- presses RAN translation and toxicity. The frontotemporal dementia (FTD)- and port, the proteasome complex, RNA bind- These results point toward a specific amyotrophic lateral sclerosis (ALS)-caus- ing, and helicases. To validate hits from role of DDX3X in repeat RNA translation. ative (GGGGCC)n repeat expansion in the initial screen, a dual luciferase cell The RNA helicase activity of DDX3X is intron 1 of C9orf72 is proposed to drive line was used in which cells express dependent on ATPase activity. To ascer- toxicity through several modes of action NanoLuciferase (NanoLuc) via RAN trans- tain whether ATPase activity was required (Balendra and Isaacs, 2018). One poten- lation as well as firefly luciferase (FLuc) via for the effect of DDX3X on DPR levels, tial mechanism is production of dipeptide canonical (AUG) translation. 48 genes wild-type DDX3X and ATPase-defective repeat proteins (DPRs) by repeat-associ- were re-tested in this secondary screen, mutant forms of DDX3X were overex- ated non-AUG (RAN) translation of the and knockdown of the RNA helicase pressed in cells. Only the wild-type expanded repeats. This non-canonical DDX3X caused the greatest change in DDX3X could lower DPR levels, suggest- translation occurs in all three reading NanoLuc levels, increasing RAN transla- ing a dependence on ATPase activity for frames, in both the sense and antisense tion by more than 2-fold while slightly modulation of RAN translation. Recombi- directions, leading to the production of reducing AUG-FLuc translation. DDX3X nant DDX3X was then used to show direct five different DPRs: poly-GA, poly-GP, is a conserved DEAD-box RNA helicase binding of repeat RNA to DDX3X and that and poly-GR from sense transcripts and shown to be involved in RNA transcrip- GGGGCC repeat RNA could stimulate poly-GP, poly-PR, and poly-AP from anti- tion, splicing, export, and translation, as DDX3X ATPase activity. sense transcripts. One possible therapeu- well as having roles in cell cycle, tumori- Following on from this cell line and tic intervention could be to reduce RAN genesis, and stress granule formation in vitro data, Cheng et al. (2019) translation. Yet, the mechanism by which (Linder and Jankowsky, 2011). Addition- next investigated whether DDX3X modu- RAN translation occurs is still not fully un- ally, loss-of-function mutations in DDX3X lation could affect RAN-translation- derstood. Targeted genetic screens have have been identified as a cause of induced toxicity in vivo. Here, they started to identify genes that regulate X-linked recessive intellectual disability utilized a Drosophila model expressing RAN translation (Goodman et al., 2019; (Snijders Blok et al., 2015). Knockdown (GGGGCC)58 in the fly eye, which causes Linsalata et al., 2019; Yamada et al., of DDX3X caused an increase in RAN- mild eye degeneration. Expression of 2019). In this issue of Neuron, Cheng translated NanoLuc signal in all three either mutant belle (the Drosophila et al. (2019) now report the first genome- sense frames; however, no change in DDX3X homolog) or belle RNAi knock- wide screen for modulators of C9orf72 antisense RAN translation was observed, down exacerbated the eye degeneration RAN translation. They first created a re- indicating specificity for sense GGGGCC phenotype and led to increased poly-GP porter cell line expressing the C9orf72 in- repeats. The increase in DPR levels was levels. These results suggest that DDX3X tronic sequence directly upstream of the also observed when in vitro transcribed may regulate RAN translation and associ- repeats, followed by (GGGGCC)70 and GGGGCC repeat-NanoLuc RNA was ated toxicity in vivo. Importantly, in addi- an eGFP tag in the poly-GA frame. Using transfected into DDX3X knockdown cells, tion to these overexpression studies, this reporter cell line, a genome-wide indicating an effect on RAN translation Cheng et al. (2019) next investigated the CRISPR-Cas9 knockout screen was car- and not transcription. Polysome fraction- effect of DDX3X on endogenous RAN ried out using fluorescence-activated ation revealed a striking change in distri- translation in C9orf72 patient cells. In cell sorting to monitor changes in poly- bution of repeat RNA, from monosomes both C9orf72 patient lymphoblasts and GA-eGFP levels. This identified 76 in controls to translating polysomes in induced pluripotent stem cells (iPSCs), enhancer and 145 suppressor genes. DDX3X siRNA-treated cells. No change levels of poly-GP were greatly increased Gene Ontology analysis identified an in the polysome distribution of the AUG- by knockdown of DDX3X. All these data enrichment in genes involved in pathways FLuc RNA was observed, and no major in various systems, including patient cells, relevant to protein translation and degra- changes in global translation were identi- point to a role for DDX3X in regulating dation: translation initiation, RNA trans- fied using puromycin incorporation. RAN translation of C9orf72 GGGGCC Neuron 104, December 4, 2019 ª 2019 Elsevier Inc. 827 Neuron Previews result is that the region immediately up- stream of the FMR1 CGG repeats is also GC rich and that DDX3X activity is required to unwind this sequence in order to allow the PIC access to the repeats. Therefore, the requirement to unwind the upstream sequence trumps the need to maintain the secondary structure of the repeats themselves. Some unwinding of the repeats is presumably needed to allow translation to proceed after initiation has occurred. Consequently, a complex balance of maintaining and resolving repeat structure may ultimately be required for effective RAN translation. What is clear is that the effects of DDX3X are both sequence and context specific. It may also be challenging to directly target DDX3X therapeutically Figure 1. Loss of DDX3X RNA Helicase Activity Increases C9orf72 Repeat RAN Translation given its diverse roles in the cell, which The (GGGGCC)n repeat expansion in C9orf72 can be translated via repeat-associated non-AUG (RAN) suggests a relatively broad range of translation in each reading frame to produce poly-GA, poly-GP, and poly-GR dipeptide repeat proteins (DPRs). The RNA helicase activity of DDX3X reduces (GGGGCC)n RAN translation. Conversely, upon loss targets, but further work will determine of DDX3X, the repeat RNA forms secondary structures that act as IRES elements and/or stall scanning whether specificity can be achieved. In ribosome complexes, leading to increased RAN translation and DPR levels. summary, the innovative genome-wide CRISPR/Cas9 screen described here repeats, as knockdown of DDX3X consis- translation in vitro using a (CGG)100 Nano- has identified new modifiers of GGGGCC tently increases DPR levels. However, for Luc assay and improved survival of pri- repeat RAN translation, which help shed there to be therapeutic relevance, the mary rat neurons expressing (CGG)100. new light on this mysterious form of un- opposite also needs to be demon- Thus, they conclude that DDX3X knock- conventional translation. strated—that overexpression of DDX3X down suppresses RAN translation and can reduce DPR levels. Cheng et al. associated toxicity. How can DDX3X REFERENCES (2019) address this question in two sys- have opposing effects on RAN translation tems. First, they report that overexpres- of C9orf72 and FMR1 expanded repeats? Balendra, R., and Isaacs, A.M. (2018). C9orf72- mediated ALS and FTD: multiple pathways to dis- sion of DDX3X decreases poly-GP in their In the first pioneering description of ease. Nat. Rev. Neurol. 14, 544–558. dual luciferase assay. Second, they show RAN translation it was shown that the sec- that DDX3X overexpression can reduce ondary structure of the repeat RNA plays Cheng, W., Wang, S., Mestre, A.A., Fu, C., Makarem, A., Xian, F., Hayes, L.R., Lopez- poly-GP in C9orf72 patient iPSC-derived an important role. Two non-mutually Gonzalez, R., Drenner, K., Jiang, J., et al. (2018). motor neurons. Impressively, this reduc- exclusive mechanisms have been sug- C9ORF72 GGGGCC repeat-associated non-AUG translation is upregulated by stress through eIF2a tion was sufficient to reduce both gested to initiate RAN translation: cap- phosphorylation. Nat. Commun. 9,51. the increased sensitivity to glutamate- dependent scanning of the pre-initiation induced cell death and the disrupted nu- complex (PIC) and cap-independent utili- Cheng, W., Wang, S., Zhang, Z., Morgens, D.W., Hayes, L.R., Lee, S., Portz, B., Xie, Y., Nguyen, cleocytoplasmic transport observed in zation of an internal ribosome entry site B.V., Haney, M.S., et al. (2019). CRISPR-Cas9 C9orf72 patient iPSC neurons. (IRES) (Cheng et al., 2018; Green et al., Screens Identify the RNA Helicase DDX3X as a It is an attractive model in which RAN 2017; Tabet et al., 2018). RNA secondary Repressor of C9ORF72 (GGGGCC)n Repeat- Associated Non-AUG Translation. Neuron 104, translation is regulated by specific factors structure is relevant for both mechanisms, this issue, 885–898. and mechanisms. This would allow for as it is known to be important for IRES- Goodman, L.D., Prudencio, M., Srinivasan, A.R., specific targeting of RAN translation mediated translation, and repeat RNA Rifai, O.M., Lee, V.M.Y., Petrucelli, L., and Bonini, in multiple repeat expansion diseases.