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| Status |
Public on Feb 05, 2021 |
| Title |
The disease-associated proteins Drosophila Nab2 and Ataxin-2 interact with shared RNAs and coregulate neuronal morphology |
| Organism |
Drosophila melanogaster |
| Experiment type |
Other
Expression profiling by high throughput sequencing
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| Summary |
Nab2 encodes a polyadenosine RNA-binding protein (RBP) with broad roles in post-transcriptional regulation, including in RNA export, poly(A) tail length control, and mRNA splicing, and loss of its human ortholog ZC3H14 gives rise to a form of autosomal recessive intellectual disability. Understanding of Nab2/ZC3H14 function in metazoan nervous systems is limited, in part, because no comprehensive identification of metazoan-Nab2-associated RNA transcripts has yet been conducted. Moreover, many Nab2/ZC3H14 functional protein partnerships likely remain unidentified. Here we present evidence that Drosophila melanogaster Nab2 interacts with the RBP Ataxin-2 (Atx2), a neuronal translational regulator, and implicate these proteins in coordinate regulation of neuronal morphology and adult viability. We then present the first high-throughput identifications of RNAs associating with Nab2 and Atx2 in Drosophila brain neurons using an RNA immunoprecipitation-sequencing (RIP-Seq) approach. Critically, the RNA interactomes of each RBP overlap. The identities of shared associated transcripts (e.g. drk, me31B, stai) and of transcripts specific to Nab2 or Atx2 (e.g. Arpc2, tea, respectively) promise insight into the neuronal functions of and interactions between each RBP. Significantly, we find Nab2 exhibits high specificity in its RNA associations in neurons in vivo, associating with only a fraction of all polyadenylated RNAs. These Nab2-associated RNAs are overrepresented for internal A-rich motifs, suggesting such sequences may partially mediate Nab2 target selection. Taken together, these data demonstrate 1)Nab2 opposingly regulates neuronal morphology and shares associated neuronal RNAs with Atx2 and 2)Drosophila Nab2 associates with a more specific subset of polyadenylated mRNAs than its polyadenosine affinity alone may suggest.
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| Overall design |
Using an RNA immunoprecipitation-sequencing (RIP-Seq) approach on total RNA isolated from adult Drosophila melanogaster heads to identify RNAs associating with Nab2 and Atx2 in Drosophila brain neurons. RNA from twelve samples of 300 adult female Drosophila heads each was isolated via the immunoprecipitation and extraction protocols described herein, generating twelve pairs of IP and Input samples, or twenty-four samples in total. These samples were composed of four biological replicates each of elav>Gal4 alone (control samples), elav>Nab2-FLAG, and elav>Atx2-3xFLAG.
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| Contributor(s) |
Rounds JC, Moberg KH |
| Citation missing |
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| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| F31 HD088043 |
Defining roles of intellectual disability protein dNab2 in neuronal translation |
EMORY UNIVERSITY |
John Christopher Rounds |
| R01 MH107305 |
Neurodevelopmental Role of an RNA Binding Protein Required for Cognitive Function |
EMORY UNIVERSITY |
Kenneth H Moberg |
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| Submission date |
Jan 27, 2021 |
| Last update date |
Feb 07, 2021 |
| Contact name |
Ken Moberg |
| E-mail(s) |
[email protected]
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| Phone |
4042177708
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| Organization name |
Emory University School of Medicine
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| Department |
Cell Biology
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| Lab |
Room 442
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| Street address |
615 Michael Street
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| City |
Atlanta |
| State/province |
Georgia |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platforms (1) |
| GPL19132 |
Illumina NextSeq 500 (Drosophila melanogaster) |
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| Samples (24)
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| Relations |
| BioProject |
PRJNA695300 |
| SRA |
SRP303538 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE165677_DESeq2_Normalized_Counts-combined_output_from_separate_RIP_and_PUT_analyses-_Parametric__Galaxy267_and_231_.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
| GSE165677_Percent_Input_Calculations_and_RBP-RNA_association_ANOVA_stat_testing.txt.gz |
2.6 Mb |
(ftp)(http) |
TXT |
| GSE165677_RAW.tar |
1.9 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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