Arginine-rich C9ORF72 ALS proteins stall ribosomes in a manner distinct from a canonical ribosome-associated quality control substrate

Abstract

Hexanucleotide expansion mutations in C9ORF72 are a frequent cause of amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPRs), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here, we explored the molecular features of DPR-induced stalling and examined whether known mechanisms such as ribosome quality control (RQC) regulate translation elongation on sequences that encode arginine-rich DPRs. We demonstrate that arginine-rich DPRs lead to stalling in a length-dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40×Gly-Xxx DPRs shows that stalling is most pronounced when Xxx is a charged amino acid (Arg, Lys, Glu, or Asp). Through a genome-wide knockout screen, we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, act differently in the case of arginine-rich DPRs. Indeed, these findings point to a limited scope for natural regulatory responses to resolve the arginine-rich DPR stalls, even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate arginine-rich DPR-mediated stalled ribosomes as a source of stress and toxicity and may be a crucial component in pathomechanisms. Hexanucleotide expansion mutations in C9ORF72 are a frequent cause of amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPRs), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here, we explored the molecular features of DPR-induced stalling and examined whether known mechanisms such as ribosome quality control (RQC) regulate translation elongation on sequences that encode arginine-rich DPRs. We demonstrate that arginine-rich DPRs lead to stalling in a length-dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40×Gly-Xxx DPRs shows that stalling is most pronounced when Xxx is a charged amino acid (Arg, Lys, Glu, or Asp). Through a genome-wide knockout screen, we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, act differently in the case of arginine-rich DPRs. Indeed, these findings point to a limited scope for natural regulatory responses to resolve the arginine-rich DPR stalls, even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate arginine-rich DPR-mediated stalled ribosomes as a source of stress and toxicity and may be a crucial component in pathomechanisms. GGGGCC hexanucleotide repeat expansion mutations in intron 1 of C9ORF72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (1DeJesus-Hernandez M. Mackenzie I.R. Boeve B.F. Boxer A.L. Baker M. Rutherford N.J. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Google Scholar, 2Renton A.E. Majounie E. Waite A. Simon-Sanchez J. Rollinson S. Gibbs J.R. et al.A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.Neuron. 2011; 72: 257-268Google Scholar). Normally, there are less than 24 repeats, whereas the length is expanded to often hundreds in ALS-causing alleles (3Van Mossevelde S. van der Zee J. Cruts M. Van Broeckhoven C. Relationship between C9orf72 repeat size and clinical phenotype.Curr. Opin. Genet. Dev. 2017; 44: 117-124Google Scholar). The expanded GGGGCC leads to the production of sense and antisense mRNA products that display two unusual features that have been postulated to contribute to disease. One is that the mRNA can form granular intracellular foci that contribute to toxicity through RNA-based mechanisms (4McEachin Z.T. Parameswaran J. Raj N. Bassell G.J. Jiang J. RNA-mediated toxicity in C9orf72 ALS and FTD.Neurobiol. Dis. 2020; 145: 105055Google Scholar). The other is that both sense and antisense mRNA can be translated through alternative initiation codons (i.e., non-AUG translation) to produce five distinct dipeptide-containing polymers (poly-GA, GR, GP, PR, and PA) (5Zu T. Liu Y. Banez-Coronel M. Reid T. Pletnikova O. Lewis J. et al.RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E4968-E4977Google Scholar). These abnormal proteins accumulate in the brain of patients with C9ORF72 mutations. The two arginine-containing dipeptide repeats (DPR), poly-GR and poly-PR, have been shown to be particularly toxic when added to cells or when expressed in cellular and organismal models (6Kwon I. Xiang S. Kato M. Wu L. Theodoropoulos P. Wang T. et al.Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.Science. 2014; 345: 1139-1145Google Scholar, 7Wen X. Tan W. Westergard T. Krishnamurthy K. Markandaiah S.S. Shi Y. et al.Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death.Neuron. 2014; 84: 1213-1225Google Scholar, 8Mizielinska S. Gronke S. Niccoli T. Ridler C.E. Clayton E.L. Devoy A. et repeat cause in through arginine-rich 2014; 345: Scholar). The toxicity is the and are encoded by that the is toxic and therefore from the mRNA X. Tan W. Westergard T. Krishnamurthy K. Markandaiah S.S. Shi Y. et al.Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death.Neuron. 2014; 84: 1213-1225Google Scholar, 8Mizielinska S. Gronke S. Niccoli T. Ridler C.E. Clayton E.L. Devoy A. et repeat cause in through arginine-rich 2014; 345: Scholar). The of mechanisms that and of translation O. quality Scholar). translation of with with or other to elongation that codons and with codons mRNA stalling for quality Dev. 2017; Scholar). in elongation on such can lead to of are for ribosome quality control (RQC) that such stalls and causes of both mRNA and a for 2020; Scholar, K. T. T. on the molecular mechanism of quality by ribosome Genet. Scholar, is for quality control 2017; Scholar, O. and of stalled quality 2020; Scholar). the stalls and can have reported that of a gene that leads to stalling on and the of a ribosome such stalls, causes in I. P. et stalling by of a causes 2014; 345: Scholar). with a of of that stalled for a of a in quality Scholar). from and that of long repeats of poly-GR and causes severe stalling M. Reid et in C9ORF72 and of 2020; Scholar, J. J. J. C. in the of and that the charged the ribosome E. S. A. S. et by and poly-GR proteins by Scholar). the mechanisms by stalling and whether to toxicity is whether known mechanisms that stalled ribosomes in cells are of and regulating the translation of poly-GR and Here, we examined the molecular features of stalling of poly-GR and We examined the length of the repeat to the of in a of DPR-mediated and a for genetic of stalling on findings that the mechanisms in stalling caused by long DPRs distinct to the mechanisms to stalling on by polyadenosine that findings point to a mechanism of toxicity that from a natural to resolve stalls in the cell of and the stalls contribute to an of find the DPR length the is long to we a of sequences coding and DPRs of and the the of DPRs. sequences to mRNA sequences and The of stalling the previously O. J. A. et al.A quality control of and translation Scholar, S. of quality control translation ribosome 2017; Scholar, E. M. J. A. and regulate quality control by regulatory 2017; Scholar, A. C. C. T. P. et and ribosome quality control of 2017; Scholar). this of a is in and with sequences and sequences the from the and and cause the ribosome to the a mRNA can proteins of gene in a 2013; Scholar). The to as a of whether the causes the in the stalling cells with the and of and by cell both and poly-GR proteins the in a length-dependent of stalled poly-GR of stalling to the repeat lengths and on both DPRs in from repeats, may toxicity or of mRNA and we these the is that is toxic in this even repeat lengths toxic longer lengths M. Reid et in C9ORF72 and of 2020; to repeats, whereas a the of repeat lengths of These findings therefore point to a stalling than repeat that other DPRs with C9ORF72 hexanucleotide expansion and to lead to stalling M. Reid et in C9ORF72 and of 2020; we examined whether stalling in a DPR to of DPRs in the stalling a DPR of amino with The sequences to and The of between and may be to in mRNA or DPR-induced The DPRs charged (Arg, Glu, and or to the in of ribosome stalling and DPRs to amino acid that the elongation of charged DPRs with to DPRs from other the charged DPRs and severe in than the charged DPRs and that charged amino acid can the stalling of translation J. C. in the elongation Scholar). The of to by for the charged amino whether stalling a in gene as the we We on the for the of in less stalling than the and a to that or We cell provides a control with with codons therefore a RQC substrate, and a for the or the component that cells to the other as as the cells that that of polyadenosine to in gene whereas to Indeed, in genes to cells genes and of these than and genes and of these genes by than the genes than One and two genes and of two than a that in the cells and that that to ribosome biogenesis, RNA RNA RNA and RNA in to been reported that the of translation to of with ribosomes and other of the translation P. M. et dipeptide repeats the and of Scholar, M. J. M. et and C9orf72 ribosomes as Sci. Scholar, Niccoli T. N. et arginine-rich dipeptide proteins with proteins in vivo to a toxic that is by Scholar). with and These are with the stalled to ribosomes or to ribosome stalls, whereas in the case of the stalls are to the ribosome may a to genes in linked to is in the with findings of the of the T. S. M. M. et and stress and with proteins M. Reid et in C9ORF72 and of 2020; Scholar). the for in M. Liu W. et repeat proteins in C9ORF72 2020; as we an upregulation of to cell such as of nuclear of of cell of chromosome and as a of expression. of to and other to cellular These findings there a to the expression. is with that and M. Reid et in C9ORF72 and of 2020; Scholar, M. J. M. et and C9orf72 ribosomes as Sci. Scholar). The other point of the upregulation of genes as as genes in stress These with the that stalling of by polyadenosine can be by proteins that in cell whereas stalled are to cell and a stress from a whether mechanisms that are known to regulate stalls on polyadenosine mRNA are in and to stalls, we a genome-wide knockout the genes a of gene N. M. M. et to and of Scholar). We two with the and the other with by codons as a for known RQC regulatory mechanisms E. M. J. A. and regulate quality control by regulatory 2017; Scholar, S. J. A. S. et of ribosome stalling translation of a Scholar, P. J. Wu O. et mechanism of stalling by and J. 2020; Scholar). to and two of cells the and of cells with the the cells with the cells with most stalling The of the that a gene translation on the or quality control to stalled the of this gene stalling (i.e., cells with this knockout be in the of genes translation on sequences be in the the of The gene in these than and gene in of these by than and The gene in of these by than and gene in of these by than gene in common for both that and there genes in common that to stalling shows a of gene for of gene that in and from the The genes in the a previously known of the RQC S. of quality control translation ribosome 2017; and of and that ribosomes that from stalling on polyadenosine mRNA S. L. The 2020; These in is with other knockout to S. of quality control translation ribosome 2017; Scholar, E. M. J. A. and regulate quality control by regulatory 2017; Scholar, S. L. The 2020; Scholar). gene in RQC E. M. J. A. and regulate quality control by regulatory 2017; in with knockout from and on the for in of these findings in the for the genes in and stalled previously shown that known as translation on K. M. et and translation initiation of to quality 2020; and is to ribosome with S. P. of translation 2020; Scholar). to screen, cells with and to have stalling with the the that in the of on polyadenosine mRNA is for genes in of stalled of gene of gene on a leads to stalling on a of gene ribosome and for and RQC S. S. is a quality control of 72: of the of RQC on ribosomes E. M. J. A. and regulate quality control by regulatory 2017; a of that ribosomes S. L. The 2020; Scholar, S. T. T. of a that quality control in 2020; a of that ribosomes S. T. T. of a that quality control in 2020; of that to of to ribosome S. T. T. of a that quality control in 2020; known as translation on K. M. et and translation initiation of to quality 2020; and is to ribosome with S. P. of translation 2020; in the of ribosomes stalled the of a mRNA and therefore mRNA in the S. L. The 2020; Scholar, by and of ribosomes and stalled elongation J. 2011; ribosome on codons I. P. et stalling by of a causes 2014; 345: Scholar, M. A.L. T. et ribosomes to neuronal 2020; of gene on a leads to stalling on a in a known to be in the RQC a distinct in the cells with and in is the to we for and in the in the known ribosome that can elongation stalled the of mRNA and by and of ribosomes and stalled elongation J. 2011; with ribosome or ribosomes stalled codons M. A.L. T. et ribosomes to neuronal 2020; in the These that and quality control mechanisms that stalled ribosomes on polyadenosine sequences or other be to this for to with genes in we both polyadenosine and poly-PR, to translation gene the and These are with the of translation with translation the in is by in that of proteins of and is for quality control 2017; Scholar). of and and are to repeats by of M. C. N. et and amino acid the of of the of stalling on on The of these genes and provides that can of translation on genes RQC in both the genes that the and quality control mechanisms of stalling is between polyadenosine and mRNA the of nuclear and nuclear genes in may to the of the to the we examined the of genes in by knockout cell These genes on in the and for and for and for both or between and and on the we a control that a known to stalling O. J. A. et al.A quality control of and translation Scholar, S. of quality control translation ribosome 2017; Scholar, E. M. J. A. and regulate quality control by regulatory 2017; Scholar, A. C. C. T. P. et and ribosome quality control of 2017; and have been previously shown to of by codons when are S. of quality control translation ribosome 2017; Scholar, E. M. J. A. and regulate quality control by regulatory 2017; Scholar). knockout the to by and for the both the for the in with the that stalling pronounced when these genes are of the that on the on control that to translation genes that on or on for of we that the of gene on are the of these are with the mechanisms in stalls on polyadenosine mRNA on elongation even may be to sense such translation as on upregulation in the of by on on the in in cell to control the on a between cell a cell control cell with cell to the on is from the on a a between cells an and cells between in in cells with an gene by the in cells as by and a of and M. in and nuclear ribosome M. M. T. A. T. M. et of and and of the for of the in M. M. I. K. K. et of RNA and with the in cell the cellular translation stress K. I. K. M. A. The stress biogenesis, to to with M. C. C. N. et of from the ribosome and ribosome Wang L. Wang Y. Shi Y. P. et is for and ribosome in J. 2020; and ribosome N. E. et from and to component of of RQC on polyadenosine mRNA E. M. J. A. and regulate quality control by regulatory 2017; for RAN translation of C9orf72 repeats Niccoli T. Shi Y. et is for RAN translation of C9orf72 and other RNA with the RNA N. M. E. Y. C. et of a nuclear for of RQC on polyadenosine mRNA S. of quality control translation ribosome 2017; on the in in the on a between cell a cell control cell with the on is from the on a a between cells an and cells between in in cells with an gene by the in cells as by and in a We examined whether these genes with stalls than of the gene on the that the stalling to that of the two that stalling for or to have on may be by the that is stalled than and to be stalled M. Reid et in C9ORF72 and of 2020; and elongation of sequences by genetic we that and for to control is a translation initiation that and from of causes a in and E. A. L. C. P. et and of that the of to 2020; Scholar). whether the ribosome and on mRNA can the elongation of we an on that the of from sequences and translation is for quality control 2017; Scholar). we of translation initiation to the of ribosomes on of cells the and of 2011; Scholar). for a mechanism of to a in for to of a on and on and The of pronounced in cells with of the findings that when the of ribosomes on mRNA transcripts is there is a for a in stalling for the DPRs. whether mechanisms are to this remains to be as to be by the known RQC findings that ribosomes long DPRs to a stalling and that the stalling mechanism by quality control The to the to is with by the in the M. J. M. et and C9orf72 ribosomes as Sci. Scholar). Whether ribosome is to ribosome as to a of remains to be by and other The findings are in with findings that DPRs are to other cellular proteins M. Reid et in C9ORF72 and of 2020; Scholar). such stalling may be mechanism of that and the of stress and have shown that ribosome stalling leads to cell M. Reid et in C9ORF72 and of 2020; this mechanism is in the of that is by a in cellular causes ribosome van der J. J. ribosome to Scholar). of charged amino acid sequences in on mechanisms to resolve or stalls, the of other may a and for toxicity in and knockout to regulatory mechanisms for regulating DPR when we translation initiation with we a in the that there are mechanisms that can the of DPR stalls or that RQC can to resolve such stalls less than for Indeed, a that a of the poly-GR in cells when J. J. J. C. in the of Scholar). this a in an ALS from a in ALS ALS and of a in amyotrophic lateral J. Genet. that to when that the mechanisms to RQC are on the 40×Gly-Xxx DPR screen, we find that elongation is in of amino to the control with and and the these from Glu, and Lys, the of that of the may a of on stalling or in the length of amino between the amino and the amino that the the charged amino (Arg, Lys, Glu, and that a of charged amino in the repeat is to translation of whether that is a or of the translation by is that this from a of S. The of in and in J. Sci. 2020; the of T. N. M. et acid with 2014; Scholar, T. and amino acid in the of and Natl. Acad. Sci. U. S. A. Scholar). The and the and the other amino is that remains is the mechanism by DPR stalls have that charged in the that the and from other the of the and the for the J. Jiang Y. C. et elongation of charged are to Scholar, Y. have a of on the of ribosome on Scholar). The ribosome can amino of a and to amino for a in an N. The ribosome as a for and of Scholar). DPRs than repeats (i.e., amino for the and to 40 repeats amino for the to cause of stalling (i.e., or lengths be to the ribosome and through an mechanism as previously M. Reid et in C9ORF72 and of 2020; Scholar, J. J. J. C. in the of Scholar, E. S. A. S. et by and poly-GR proteins by Scholar). longer dipeptide lengths repeats for poly-GR amino and repeats for amino are to than The of sequences that encode poly-GR or by the ribosome in a length-dependent that DPR is in may be of a of by the of that the of and translation in vitro and the of ribosomes E. S. A. S. et by and poly-GR proteins by Scholar). is to that DPR poly-GR or bind and the of ribosomes on the mRNA to of translation in and a of The to this mechanism from poly-GR and with proteins M. Reid et in C9ORF72 and of 2020; Scholar, M. J. M. et and C9orf72 ribosomes as Sci. and to be with to this mechanism from that the DPRs can a length of repeats van der E. of toxic dipeptide repeat proteins to C9orf72 J. 2020; Scholar). translation may be through a of ribosomes and of a of toxic stress to cellular to is the poly-GR to be stalling than One is that the than van der E. of toxic dipeptide repeat proteins to C9orf72 J. 2020; Scholar). One is that may to accumulate in the ribosome may through Indeed, the is to to the ribosome J. S. L. M. et the ribosome Scholar). may to in ribosomes in a from cells to genes in translation and of these responses from the toxicity previously from poly-PR, of translation P. M. et dipeptide repeats the and of Scholar, M. J. M. et and C9orf72 ribosomes as Sci. Scholar, Niccoli T. N. et arginine-rich dipeptide proteins with proteins in vivo to a toxic that is by and the of M. Reid et in C9ORF72 and of 2020; Scholar, T. S. M. M. et and stress Scholar, L. S. M. A. et arginine-containing dipeptide repeats with and impede Scholar). responses to stress may be to of the of RQC genes that cellular mechanisms to the of DPR stalled even these mechanisms may be to other to a for RQC the of poly-GR by the the J. J. J. C. in the of Scholar). the stalling may be or by that to with such stalling is that the other stress responses are in to that to resolve through the are DPR in brain to with of the P. van I. S. W. et in C9orf72 frontotemporal dementia is with dipeptide and in Scholar). for the DPRs a mechanism of stalling that cellular the to for coding the and in of amino acid the of this as in as M. Reid et in C9ORF72 and of 2020; Scholar). to a repeat of and to the for and PR, and GP, and as S. et and 1 distinct in the 2014; Scholar). knockout cell sequences genes of from O. and genome-wide for 2014; as in the from cell and by and and for of or or sequences added to the of by with cells The is from the in with and in a with the in added to cells a of culture and cells by 24 cells with and 24 and and to for cell with the and to for with the and and and in and and to to the for control as by for and in for and for by and on by The with in for 1 by an in or in for 1 The in and with in for 1 by an for knockout knockout cell and cell with cells with from et gene by in from and in and cells with in the of The with 24 for cells with 1 in for we cell and a by 1 in in of added to in the by and for the of cells with and in 1 and with a cell cells in to the for on cells through a and for The RNA in the RNA RNA by RNA from of The mRNA mRNA with on the by the for and the to the in as in C. O. J.R. et mRNA translation to of ribosome J. 2020; Scholar). cells with or with of for to by and by on of to for for an The with of by an for by of on and for 1 The in in for 1 by an in in for 1 The in and with in for 1 by an in to and of and in for to a with Scholar). gene W. S. of and for with 2014; in The between the of and with as a A. P. S. et a for genome-wide Natl. Acad. Sci. U. S. A. Scholar, A. S. J. et genes in are in Genet. of genes with by for the gene from the A. C. M. P. The gene Scholar). with as in for with P. A. O. Wang et a for models of Scholar). from the are The knockout from from and N. M. M. et to and of Scholar). The as in the J. S. et knockout and 2017; with of cells of of cells with or the cells to the other as the with a cell for the and of the and from cells with as previously S. K. O. K. Shi X. et in a of and Scholar). The by as previously M. C. et for genetic with Scholar). The with the by the in between the control W. T. L. et of genes from knockout 2014; and for gene genes with and are in of the or as of for on in and to stalling in of on in and from the in and in L. in for from the are and production with the as than knockout for by T. N. J. T. Waite K. et of from and the region of gene of The sequences gene are shown in a as a in for or gene knockout by the from cells to from cells knockout in on a in on a that cause of of are in the and in are as in The to in between of DPRs other the of of to control in and the in and gene of the of and from for cell from on with than cells for the and to control as RNA from this have been to with the from the of and knockout screening are in and the findings of this are from the The that have of with the of this L. and M. A. E. P. E. S. L. M. E. and L. K. K. A. and E. P. K. L. and M. K. and M. A. and E. P. K. E. M. E. J. S. L. M. E. L. and M. J. L. and M. S. L. M. E. and E. M. M. M. by to M. and and and to L. of and through the

Topics & Concepts