RNA-protein interactions are crucial for regulating various cellular processes such as gene expression,RNA modification and translation.In contrast,undesirable RNA-protein interactions often cause dysregulated cellula...RNA-protein interactions are crucial for regulating various cellular processes such as gene expression,RNA modification and translation.In contrast,undesirable RNA-protein interactions often cause dysregulated cellular activities associated with many human diseases.The RNA containing expanded GGGGCC repeats forms secondary structures that sequester various RNA binding proteins(RBPs),leading to the development of amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD).However,a gap persists in understanding the structural basis for GGGGCC repeat RNA binding to RBPs.Here,we resolve the first solution NMR structure of a natural GGGGCC repeat RNA containing a 2×2 GG/GG internal loop,and perform MD simulations and site-directed mutagenesis to elucidate the mechanism for GGGGCC repeat RNA binding to SRSF_(2),a splicing factor and key marker of nuclear speckles.We reveal that the R47/T51/R61 residues in RNA recognition motif of SRSF_(2) and the 2×2 GG/GG internal loop in GGGGCC repeat RNA are essential for binding.This work furnishes a valuable high-resolution structural basis for understanding the binding mechanism for GGGGCC repeat RNA and RBPs,and steers RNA structure-based drug design.展开更多
A largely increased number of GGGGCC repeats located in the non-coding region of C9orf72 gene have been identified as the leading cause of two related neurological disorders, familial amyotrophic lateral sclero- sis ...A largely increased number of GGGGCC repeats located in the non-coding region of C9orf72 gene have been identified as the leading cause of two related neurological disorders, familial amyotrophic lateral sclero- sis （ALS） and frontotemporal dementia （FTD）. We examined G-quadruplex forming ability of GGGGCC- repeat containing oligonucleotides with four guanine tracts chosen as the smallest possible model to form a unimolecular G-quadruplex. These oligonucleotides are readily to folded into G-quadruplexes in the presence of K~ ions. However, the formation of multiple structures makes structural analysis challenging and time consuming. We observed that flanking sequences on 5＇- and 3＇-ends as well as mutations of loop residues have a profound effect on folding. Sequence d[（GaC2）3G4] was chosen for further scrutiny and optimization of nuclear magnetic resonance （NMR） spectroscopic properties with dG to 8Br-dG substitutions at specific positions in the sequence under different folding conditions. Expectedly, folding into desired predominant topology is facilitated when sub- stituted residue adopted a syn conformation in the naturally-occurring structure. Single dG to 8Br-dG sub- stitution at position 21 and fine tuning of folding conditions facilitate folding of d[（G4C2）3GG^BrGG] into （mostly） a single G-quadruplex, and thus enable determi- nation of its high-resolution structure by high-field NMR.展开更多
Background:The GGGGCC(G4C2)repeat expansion in the human open reading frame 72 on chromosome 9,C9orf72,is the most common cause of amyotrophic lateral sclerosis(ALS).Studies in transgenic mouse models have linked the ...Background:The GGGGCC(G4C2)repeat expansion in the human open reading frame 72 on chromosome 9,C9orf72,is the most common cause of amyotrophic lateral sclerosis(ALS).Studies in transgenic mouse models have linked the pathogenic mechanism of G4C2 repeat expansion to RNA foci or the accumulation of unnatural dipeptide repeats in neurons.However,only one of the existing transgenic mouse lines developed typical ALS.Methods:C9orf72 knockin rats were generated by knockin of 80 G4C2 repeats with human flanking fragments within exon1a and exon1b at the rat C9orf72 locus.Protein expression was detected by western blot.Motor coordination and grip force were measured using a Rotarod test and a grip strength test.Neurodegeneration was assessed by Nissl staining with cresyl violet.Results:C9orf72 haploinsufficiency reduced C9orf72 protein expression 40%in the cerebrum,cerebellum and spinal cords from knockin rats(P<.05).The knockin(KI)rats developed motor deficits from 4 months of age.Their falling latencies and grip force were decreased by 67%(P<.01)and 44%(P<.01),respectively,at 12 months of age compared to wild-type(WT)mice.The knockin of the hexanucleotide repeat expansion(HRE)caused a 47%loss of motor neurons in the spinal cord(P<.001)and 25%(5/20)of female KI rats developed hind limb paralysis at 13 to 24 months.Conclusion:Motor defects in KI rats may result from neurotoxicity caused by HRE and the resulting reduction in C9orf72 protein due to haploinsufficiency.These KI rats could be a useful model for investigating the contributions of loss-of-function to neurotoxicity in C9orf72-related ALS.展开更多
基金supported by the National Key Research and Development Program of China(2021YFA0909400)National Natural Science Foundation of China(22374132,22225402,32341017)+1 种基金Department of Science and Technology of Zhejiang Province(2024R01005,2023SDYXS0002)Natural Science Foundation of Zhejiang Province(QKHM25B0501).
文摘RNA-protein interactions are crucial for regulating various cellular processes such as gene expression,RNA modification and translation.In contrast,undesirable RNA-protein interactions often cause dysregulated cellular activities associated with many human diseases.The RNA containing expanded GGGGCC repeats forms secondary structures that sequester various RNA binding proteins(RBPs),leading to the development of amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD).However,a gap persists in understanding the structural basis for GGGGCC repeat RNA binding to RBPs.Here,we resolve the first solution NMR structure of a natural GGGGCC repeat RNA containing a 2×2 GG/GG internal loop,and perform MD simulations and site-directed mutagenesis to elucidate the mechanism for GGGGCC repeat RNA binding to SRSF_(2),a splicing factor and key marker of nuclear speckles.We reveal that the R47/T51/R61 residues in RNA recognition motif of SRSF_(2) and the 2×2 GG/GG internal loop in GGGGCC repeat RNA are essential for binding.This work furnishes a valuable high-resolution structural basis for understanding the binding mechanism for GGGGCC repeat RNA and RBPs,and steers RNA structure-based drug design.
文摘A largely increased number of GGGGCC repeats located in the non-coding region of C9orf72 gene have been identified as the leading cause of two related neurological disorders, familial amyotrophic lateral sclero- sis （ALS） and frontotemporal dementia （FTD）. We examined G-quadruplex forming ability of GGGGCC- repeat containing oligonucleotides with four guanine tracts chosen as the smallest possible model to form a unimolecular G-quadruplex. These oligonucleotides are readily to folded into G-quadruplexes in the presence of K~ ions. However, the formation of multiple structures makes structural analysis challenging and time consuming. We observed that flanking sequences on 5＇- and 3＇-ends as well as mutations of loop residues have a profound effect on folding. Sequence d[（GaC2）3G4] was chosen for further scrutiny and optimization of nuclear magnetic resonance （NMR） spectroscopic properties with dG to 8Br-dG substitutions at specific positions in the sequence under different folding conditions. Expectedly, folding into desired predominant topology is facilitated when sub- stituted residue adopted a syn conformation in the naturally-occurring structure. Single dG to 8Br-dG sub- stitution at position 21 and fine tuning of folding conditions facilitate folding of d[（G4C2）3GG^BrGG] into （mostly） a single G-quadruplex, and thus enable determi- nation of its high-resolution structure by high-field NMR.
基金National Natural Science Foundation of China(81571222),CAMS Innovation Fund for Medical Sciences(CIFMS,2016-I2M-1-004)Beijing Municipal Natural Science Foundation(7172135)。
文摘Background:The GGGGCC(G4C2)repeat expansion in the human open reading frame 72 on chromosome 9,C9orf72,is the most common cause of amyotrophic lateral sclerosis(ALS).Studies in transgenic mouse models have linked the pathogenic mechanism of G4C2 repeat expansion to RNA foci or the accumulation of unnatural dipeptide repeats in neurons.However,only one of the existing transgenic mouse lines developed typical ALS.Methods:C9orf72 knockin rats were generated by knockin of 80 G4C2 repeats with human flanking fragments within exon1a and exon1b at the rat C9orf72 locus.Protein expression was detected by western blot.Motor coordination and grip force were measured using a Rotarod test and a grip strength test.Neurodegeneration was assessed by Nissl staining with cresyl violet.Results:C9orf72 haploinsufficiency reduced C9orf72 protein expression 40%in the cerebrum,cerebellum and spinal cords from knockin rats(P<.05).The knockin(KI)rats developed motor deficits from 4 months of age.Their falling latencies and grip force were decreased by 67%(P<.01)and 44%(P<.01),respectively,at 12 months of age compared to wild-type(WT)mice.The knockin of the hexanucleotide repeat expansion(HRE)caused a 47%loss of motor neurons in the spinal cord(P<.001)and 25%(5/20)of female KI rats developed hind limb paralysis at 13 to 24 months.Conclusion:Motor defects in KI rats may result from neurotoxicity caused by HRE and the resulting reduction in C9orf72 protein due to haploinsufficiency.These KI rats could be a useful model for investigating the contributions of loss-of-function to neurotoxicity in C9orf72-related ALS.
