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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 08, 2023 |
| Title |
A Massively Parallel Screen of 5′UTR Mutations Identifies Variants Impacting Translation and Protein Production in Neurodevelopmental Disorder Genes |
| Organisms |
Homo sapiens; Mus musculus |
| Experiment type |
Other
|
| Summary |
De novo mutations cause a variety of neurodevelopmental disorders including autism. Recent whole genome sequencing has identified hundreds of mutations in untranslated regions (UTRs) of genes from individuals with autism, but it is impossible to predict from sequence alone which are functional, and thus might be causal. Therefore, we developed a high throughput assay to screen the consequence over 1,000 variants from 5'UTRs mutations on transcript abundance and translation efficiency. This assay successfully enriched for elements that alter reporter translation, identifying over 100 potentially functional mutations. Studies in patient-derived cell lines further confirmed these mutations alter protein production in individuals in autism, including for multiple genes known to cause of syndromic forms autism, suggesting a diagnosis for these individual patients. Since UTR function varies by cell type, we further optimized this high throughput assay to enable assessment of mutations in neurons of the living brain. Neurons demonstrate profoundly different principles of regulation by 5'UTRs, consistent with more robust mechanism for reducing impact of 5'UTR RNA structure. Overall our results highlight a new approach for assessing the impact of 5’UTR across cell types and suggest some cases of neurodevelopmental disorder may be caused by such variants.
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| Overall design |
Pooled library screens of synthetic reporter genes containing variable 5' UTR sequences and associated barcode sequences in the 3'UTR were performed either in HEK cells or in mouse brain in vivo in order to measure the effects of 5'UTR elements and their variants on transcript abundance and translation efficiency. HEK cells were transfected with a plasmid library containing approximately 33,000 uniquely barcoded reporters, and in vivo experiments were performed by transcranial delivery of AAV-packaged sub-libraries each containing a third of the total library. RNA and DNA were extracted from each sample, and RNA was subjected to density centrifugation and fraction to isolate mRNA associated with the 43S preinitation complex, monosomes, or polysomes. The barcode-containing 3'UTRs were amplified from each fraction to determine the abundance of each respective 5'UTR-associated reporter. All in vivo mouse experiments were performed in a Vglut2-Cre line so that a LoxP-flanked cassette in the 3'UTR is inverted only in glutamatergic neurons. For each in vivo RNA sample, two reverse transcription primers, one specific to the unrecombined state ("CreOFF") and one specific to the inverted state ("CreON"), were used to prepare libraries to esimtate the reporter abundance in both Cre-expressing glutamatergic neurons and non-Cre-expressing cells. For both HEK and in vivo experiments, six biological replicates were collected.
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| Web link |
https://doi.org/10.1101/2023.11.02.23297961
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| Contributor(s) |
Plassmeyer SP, Florian CP, Kasper MJ, Chase R, Mueller S, McFarland White K, Jungers CF, Pavlovic Djuranovic S, Djuranovic S, Dougherty JD |
| Citation(s) |
37961498 |
| |
| Submission date |
Oct 26, 2023 |
| Last update date |
Nov 11, 2024 |
| Contact name |
Joseph D Dougherty |
| E-mail(s) |
jdougherty@wustl.edu
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| Organization name |
Washington University in St. Louis
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| Department |
Genetics
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| Street address |
4370 Duncan Ave
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| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platforms (2) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (186)
|
| GSM7867914 |
HEK, Replicate 4, DNA |
| GSM7867915 |
HEK, Replicate 5, DNA |
| GSM7867916 |
HEK, Replicate 6, DNA |
| GSM7867917 |
HEK, Replicate 1, TotalRNA |
| GSM7867918 |
HEK, Replicate 2, TotalRNA |
| GSM7867919 |
HEK, Replicate 3, TotalRNA |
| GSM7867920 |
HEK, Replicate 4, TotalRNA |
| GSM7867921 |
HEK, Replicate 5, TotalRNA |
| GSM7867922 |
HEK, Replicate 6, TotalRNA |
| GSM7867923 |
HEK, Replicate 1, 80S_RNA |
| GSM7867924 |
HEK, Replicate 2, 80S_RNA |
| GSM7867925 |
HEK, Replicate 3, 80S_RNA |
| GSM7867926 |
HEK, Replicate 4, 80S_RNA |
| GSM7867927 |
HEK, Replicate 5, 80S_RNA |
| GSM7867928 |
HEK, Replicate 6, 80S_RNA |
| GSM7867929 |
HEK, Replicate 1, 40S_RNA |
| GSM7867930 |
HEK, Replicate 2, 40S_RNA |
| GSM7867931 |
HEK, Replicate 3, 40S_RNA |
| GSM7867932 |
HEK, Replicate 4, 40S_RNA |
| GSM7867933 |
HEK, Replicate 5, 40S_RNA |
| GSM7867934 |
HEK, Replicate 6, 40S_RNA |
| GSM7867935 |
HEK, Replicate 1, Polysome_RNA |
| GSM7867936 |
HEK, Replicate 2, Polysome_RNA |
| GSM7867937 |
HEK, Replicate 3, Polysome_RNA |
| GSM7867938 |
HEK, Replicate 4, Polysome_RNA |
| GSM7867939 |
HEK, Replicate 5, Polysome_RNA |
| GSM7867940 |
HEK, Replicate 6, Polysome_RNA |
| GSM7867941 |
HEK, Replicate 1, TRAP_RNA |
| GSM7867942 |
HEK, Replicate 2, TRAP_RNA |
| GSM7867943 |
HEK, Replicate 3, TRAP_RNA |
| GSM7867944 |
HEK, Replicate 4, TRAP_RNA |
| GSM7867945 |
HEK, Replicate 5, TRAP_RNA |
| GSM7867946 |
HEK, Replicate 6, TRAP_RNA |
| GSM7867947 |
HEK, Replicate 1, Plasmid_Library |
| GSM7867948 |
HEK, Replicate 2, Plasmid_Library |
| GSM7867949 |
HEK, Replicate 3, Plasmid_Library |
| GSM7867950 |
HEK, Replicate 4, Plasmid_Library |
| GSM7867951 |
HEK, Replicate 5, Plasmid_Library |
| GSM7867952 |
HEK, Replicate 6, Plasmid_Library |
| GSM7867953 |
Vglut, Sublibrary 1, Replicate 1, polysome RNA, CreOFF |
| GSM7867954 |
Vglut, Sublibrary 1, Replicate 1, total RNA, CreOFF |
| GSM7867955 |
Vglut, Sublibrary 1, Replicate 1, 80S RNA, CreOFF |
| GSM7867956 |
Vglut, Sublibrary 1, Replicate 1, DNA, CreOFF |
| GSM7867957 |
Vglut, Sublibrary 1, Replicate 2, total RNA, CreOFF |
| GSM7867958 |
Vglut, Sublibrary 1, Replicate 2, 80S RNA, CreOFF |
| GSM7867959 |
Vglut, Sublibrary 1, Replicate 2, polysome RNA, CreOFF |
| GSM7867960 |
Vglut, Sublibrary 1, Replicate 2, DNA, CreOFF |
| GSM7867961 |
Vglut, Sublibrary 1, Replicate 3, DNA, CreOFF |
| GSM7867962 |
Vglut, Sublibrary 1, Replicate 3, 80S RNA, CreOFF |
| GSM7867963 |
Vglut, Sublibrary 1, Replicate 3, total RNA, CreOFF |
| GSM7867964 |
Vglut, Sublibrary 1, Replicate 3, polysome RNA, CreOFF |
| GSM7867965 |
Vglut, Sublibrary 1, Replicate 4, total RNA, CreOFF |
| GSM7867966 |
Vglut, Sublibrary 1, Replicate 4, polysome RNA, CreOFF |
| GSM7867967 |
Vglut, Sublibrary 1, Replicate 4, DNA, CreOFF |
| GSM7867968 |
Vglut, Sublibrary 1, Replicate 4, 80S RNA, CreOFF |
| GSM7867969 |
Vglut, Sublibrary 2, Replicate 1, DNA, CreOFF |
| GSM7867970 |
Vglut, Sublibrary 2, Replicate 1, polysome RNA, CreOFF |
| GSM7867971 |
Vglut, Sublibrary 2, Replicate 1, total RNA, CreOFF |
| GSM7867972 |
Vglut, Sublibrary 2, Replicate 1, 80S RNA, CreOFF |
| GSM7867973 |
Vglut, Sublibrary 2, Replicate 2, polysome RNA, CreOFF |
| GSM7867974 |
Vglut, Sublibrary 2, Replicate 2, total RNA, CreOFF |
| GSM7867975 |
Vglut, Sublibrary 2, Replicate 2, 80S RNA, CreOFF |
| GSM7867976 |
Vglut, Sublibrary 2, Replicate 2, DNA, CreOFF |
| GSM7867977 |
Vglut, Sublibrary 2, Replicate 3, total RNA, CreOFF |
| GSM7867978 |
Vglut, Sublibrary 2, Replicate 3, polysome RNA, CreOFF |
| GSM7867979 |
Vglut, Sublibrary 2, Replicate 3, 80S RNA, CreOFF |
| GSM7867980 |
Vglut, Sublibrary 2, Replicate 3, DNA, CreOFF |
| GSM7867981 |
Vglut, Sublibrary 3, Replicate 1, polysome RNA, CreOFF |
| GSM7867982 |
Vglut, Sublibrary 3, Replicate 1, DNA, CreOFF |
| GSM7867983 |
Vglut, Sublibrary 3, Replicate 1, 80S RNA, CreOFF |
| GSM7867984 |
Vglut, Sublibrary 3, Replicate 1, total RNA, CreOFF |
| GSM7867985 |
Vglut, Sublibrary 3, Replicate 2, 80S RNA, CreOFF |
| GSM7867986 |
Vglut, Sublibrary 3, Replicate 2, polysome RNA, CreOFF |
| GSM7867987 |
Vglut, Sublibrary 3, Replicate 2, total RNA, CreOFF |
| GSM7867988 |
Vglut, Sublibrary 3, Replicate 2, DNA, CreOFF |
| GSM7867989 |
Vglut, Sublibrary 3, Replicate 3, polysome RNA, CreOFF |
| GSM7867990 |
Vglut, Sublibrary 3, Replicate 3, 80S RNA, CreOFF |
| GSM7867991 |
Vglut, Sublibrary 3, Replicate 3, total RNA, CreOFF |
| GSM7867992 |
Vglut, Sublibrary 3, Replicate 3, DNA, CreOFF |
| GSM7867993 |
Vglut, Sublibrary 1, Replicate 5, DNA, CreOFF |
| GSM7867994 |
Vglut, Sublibrary 1, Replicate 5, total RNA, CreOFF |
| GSM7867995 |
Vglut, Sublibrary 1, Replicate 5, 80S RNA, CreOFF |
| GSM7867996 |
Vglut, Sublibrary 1, Replicate 5, polysome RNA, CreOFF |
| GSM7867997 |
Vglut, Sublibrary 1, Replicate 6, DNA, CreOFF |
| GSM7867998 |
Vglut, Sublibrary 1, Replicate 6, 80S RNA, CreOFF |
| GSM7867999 |
Vglut, Sublibrary 1, Replicate 6, polysome RNA, CreOFF |
| GSM7868000 |
Vglut, Sublibrary 1, Replicate 6, total RNA, CreOFF |
| GSM7868001 |
Vglut, Sublibrary 2, Replicate 4, 80S RNA, CreOFF |
| GSM7868002 |
Vglut, Sublibrary 2, Replicate 4, total RNA, CreOFF |
| GSM7868003 |
Vglut, Sublibrary 2, Replicate 4, DNA, CreOFF |
| GSM7868004 |
Vglut, Sublibrary 2, Replicate 4, polysome RNA, CreOFF |
| GSM7868005 |
Vglut, Sublibrary 2, Replicate 5, 80S RNA, CreOFF |
| GSM7868006 |
Vglut, Sublibrary 2, Replicate 5, polysome RNA, CreOFF |
| GSM7868007 |
Vglut, Sublibrary 2, Replicate 5, DNA, CreOFF |
| GSM7868008 |
Vglut, Sublibrary 2, Replicate 5, total RNA, CreOFF |
| GSM7868009 |
Vglut, Sublibrary 2, Replicate 6, total RNA, CreOFF |
| GSM7868010 |
Vglut, Sublibrary 2, Replicate 6, 80S RNA, CreOFF |
| GSM7868011 |
Vglut, Sublibrary 2, Replicate 6, DNA, CreOFF |
| GSM7868012 |
Vglut, Sublibrary 2, Replicate 6, polysome RNA, CreOFF |
| GSM7868013 |
Vglut, Sublibrary 3, Replicate 4, total RNA, CreOFF |
| GSM7868014 |
Vglut, Sublibrary 3, Replicate 4, 80S RNA, CreOFF |
| GSM7868015 |
Vglut, Sublibrary 3, Replicate 4, DNA, CreOFF |
| GSM7868016 |
Vglut, Sublibrary 3, Replicate 4, polysome RNA, CreOFF |
| GSM7868017 |
Vglut, Sublibrary 3, Replicate 5, 80S RNA, CreOFF |
| GSM7868018 |
Vglut, Sublibrary 3, Replicate 5, polysome RNA, CreOFF |
| GSM7868019 |
Vglut, Sublibrary 3, Replicate 5, total RNA, CreOFF |
| GSM7868020 |
Vglut, Sublibrary 3, Replicate 5, DNA, CreOFF |
| GSM7868021 |
Vglut, Sublibrary 3, Replicate 6, polysome RNA, CreOFF |
| GSM7868022 |
Vglut, Sublibrary 3, Replicate 6, total RNA, CreOFF |
| GSM7868023 |
Vglut, Sublibrary 3, Replicate 6, 80S RNA, CreOFF |
| GSM7868024 |
Vglut, Sublibrary 3, Replicate 6, DNA, CreOFF |
| GSM7868025 |
Vglut, Sublibrary 1, Replicate 1, 80S RNA, CreON |
| GSM7868026 |
Vglut, Sublibrary 1, Replicate 1, DNA, CreON |
| GSM7868027 |
Vglut, Sublibrary 1, Replicate 1, total RNA, CreON |
| GSM7868028 |
Vglut, Sublibrary 1, Replicate 1, polysome RNA, CreON |
| GSM7868029 |
Vglut, Sublibrary 1, Replicate 2, total RNA, CreON |
| GSM7868030 |
Vglut, Sublibrary 1, Replicate 2, DNA, CreON |
| GSM7868031 |
Vglut, Sublibrary 1, Replicate 2, polysome RNA, CreON |
| GSM7868032 |
Vglut, Sublibrary 1, Replicate 2, 80S RNA, CreON |
| GSM7868033 |
Vglut, Sublibrary 1, Replicate 3, polysome RNA, CreON |
| GSM7868034 |
Vglut, Sublibrary 1, Replicate 3, total RNA, CreON |
| GSM7868035 |
Vglut, Sublibrary 1, Replicate 3, 80S RNA, CreON |
| GSM7868036 |
Vglut, Sublibrary 1, Replicate 3, DNA, CreON |
| GSM7868037 |
Vglut, Sublibrary 1, Replicate 4, total RNA, CreON |
| GSM7868038 |
Vglut, Sublibrary 1, Replicate 4, DNA, CreON |
| GSM7868039 |
Vglut, Sublibrary 1, Replicate 4, polysome RNA, CreON |
| GSM7868040 |
Vglut, Sublibrary 1, Replicate 4, 80S RNA, CreON |
| GSM7868041 |
Vglut, Sublibrary 2, Replicate 1, polysome RNA, CreON |
| GSM7868042 |
Vglut, Sublibrary 2, Replicate 1, DNA, CreON |
| GSM7868043 |
Vglut, Sublibrary 2, Replicate 1, 80S RNA, CreON |
| GSM7868044 |
Vglut, Sublibrary 2, Replicate 1, total RNA, CreON |
| GSM7868045 |
Vglut, Sublibrary 2, Replicate 2, polysome RNA, CreON |
| GSM7868046 |
Vglut, Sublibrary 2, Replicate 2, total RNA, CreON |
| GSM7868047 |
Vglut, Sublibrary 2, Replicate 2, 80S RNA, CreON |
| GSM7868048 |
Vglut, Sublibrary 2, Replicate 2, DNA, CreON |
| GSM7868049 |
Vglut, Sublibrary 2, Replicate 3, polysome RNA, CreON |
| GSM7868050 |
Vglut, Sublibrary 2, Replicate 3, 80S RNA, CreON |
| GSM7868051 |
Vglut, Sublibrary 2, Replicate 3, DNA, CreON |
| GSM7868052 |
Vglut, Sublibrary 2, Replicate 3, total RNA, CreON |
| GSM7868053 |
Vglut, Sublibrary 3, Replicate 1, total RNA, CreON |
| GSM7868054 |
Vglut, Sublibrary 3, Replicate 1, DNA, CreON |
| GSM7868055 |
Vglut, Sublibrary 3, Replicate 1, 80S RNA, CreON |
| GSM7868056 |
Vglut, Sublibrary 3, Replicate 1, polysome RNA, CreON |
| GSM7868057 |
Vglut, Sublibrary 3, Replicate 2, polysome RNA, CreON |
| GSM7868058 |
Vglut, Sublibrary 3, Replicate 2, DNA, CreON |
| GSM7868059 |
Vglut, Sublibrary 3, Replicate 2, total RNA, CreON |
| GSM7868060 |
Vglut, Sublibrary 3, Replicate 2, 80S RNA, CreON |
| GSM7868061 |
Vglut, Sublibrary 3, Replicate 3, DNA, CreON |
| GSM7868062 |
Vglut, Sublibrary 3, Replicate 3, 80S RNA, CreON |
| GSM7868063 |
Vglut, Sublibrary 3, Replicate 3, total RNA, CreON |
| GSM7868064 |
Vglut, Sublibrary 3, Replicate 3, polysome RNA, CreON |
| GSM7868065 |
Vglut, Sublibrary 1, Replicate 5, total RNA, CreON |
| GSM7868066 |
Vglut, Sublibrary 1, Replicate 5, polysome RNA, CreON |
| GSM7868067 |
Vglut, Sublibrary 1, Replicate 5, DNA, CreON |
| GSM7868068 |
Vglut, Sublibrary 1, Replicate 5, 80S RNA, CreON |
| GSM7868069 |
Vglut, Sublibrary 1, Replicate 6, polysome RNA, CreON |
| GSM7868070 |
Vglut, Sublibrary 1, Replicate 6, total RNA, CreON |
| GSM7868071 |
Vglut, Sublibrary 1, Replicate 6, DNA, CreON |
| GSM7868072 |
Vglut, Sublibrary 1, Replicate 6, 80S RNA, CreON |
| GSM7868073 |
Vglut, Sublibrary 2, Replicate 4, total RNA, CreON |
| GSM7868074 |
Vglut, Sublibrary 2, Replicate 4, polysome RNA, CreON |
| GSM7868075 |
Vglut, Sublibrary 2, Replicate 4, DNA, CreON |
| GSM7868076 |
Vglut, Sublibrary 2, Replicate 4, 80S RNA, CreON |
| GSM7868077 |
Vglut, Sublibrary 2, Replicate 5, 80S RNA, CreON |
| GSM7868078 |
Vglut, Sublibrary 2, Replicate 5, DNA, CreON |
| GSM7868079 |
Vglut, Sublibrary 2, Replicate 5, total RNA, CreON |
| GSM7868080 |
Vglut, Sublibrary 2, Replicate 5, polysome RNA, CreON |
| GSM7868081 |
Vglut, Sublibrary 2, Replicate 6, 80S RNA, CreON |
| GSM7868082 |
Vglut, Sublibrary 2, Replicate 6, total RNA, CreON |
| GSM7868083 |
Vglut, Sublibrary 2, Replicate 6, polysome RNA, CreON |
| GSM7868084 |
Vglut, Sublibrary 2, Replicate 6, DNA, CreON |
| GSM7868085 |
Vglut, Sublibrary 3, Replicate 4, total RNA, CreON |
| GSM7868086 |
Vglut, Sublibrary 3, Replicate 4, 80S RNA, CreON |
| GSM7868087 |
Vglut, Sublibrary 3, Replicate 4, polysome RNA, CreON |
| GSM7868088 |
Vglut, Sublibrary 3, Replicate 4, DNA, CreON |
| GSM7868089 |
Vglut, Sublibrary 3, Replicate 5, total RNA, CreON |
| GSM7868090 |
Vglut, Sublibrary 3, Replicate 5, DNA, CreON |
| GSM7868091 |
Vglut, Sublibrary 3, Replicate 5, polysome RNA, CreON |
| GSM7868092 |
Vglut, Sublibrary 3, Replicate 5, 80S RNA, CreON |
| GSM7868093 |
Vglut, Sublibrary 3, Replicate 6, polysome RNA, CreON |
| GSM7868094 |
Vglut, Sublibrary 3, Replicate 6, total RNA, CreON |
| GSM7868095 |
Vglut, Sublibrary 3, Replicate 6, 80S RNA, CreON |
| GSM7868096 |
Vglut, Sublibrary 3, Replicate 6, DNA, CreON |
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| Relations |
| BioProject |
PRJNA1032859 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE246381_hek_combined_umi_counts.csv.gz |
2.6 Mb |
(ftp)(http) |
CSV |
| GSE246381_vglut_combined_umi_counts.csv.gz |
4.3 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
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