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| Status |
Public on Jul 09, 2022 |
| Title |
Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
RNA transport and local translation provide spatial control of gene expression, and RNA binding proteins (RBPs) act as critical adapters in this multi-step process. Muscleblind-like (MBNL) RNA binding proteins, implicated in Myotonic Dystrophy and cancer, localize RNAs to myoblast membranes and distal neurites, through unknown mechanisms. We found that MBNLs form motile and anchored granules in neurons and myoblasts, and selectively associate with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs also associate with these kinesins, implicating a motor-RBP specificity code. Live cell imaging and fractionation revealed that membrane anchoring is mediated through the unstructured carboxy-terminal tail of MBNL1. Both kinesin- and membrane-recruitment functions were reconstituted using MBNL-MS2 coat protein fusions; this approach, termed RBP Module Recruitment and Imaging (RBP-MRI), decouples RNA binding, kinesin recruitment, and membrane anchoring functions, while also establishing general strategies for studying multi-functional, modular domains of RBPs.
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| Overall design |
The underside of hanging cell culture inserts with 1 µm pores (Corning, 353102) were coated with diluted Matrigel preparation (Corning, 354277) and placed into specialized, deep 6-well plates (Corning, 353502). 2 mL of 10% DMEM media was placed on top and underneath the hanging insert membrane prior to cell plating. Neuro2a cells were then densely plated onto membranes at a concentration of 1✕10^6. After 24 hours, serum-containing media was replaced with no-serum media and neurites were allowed to grow for 48 hours. Subsequently, no serum media was removed from both sides of the membrane, which was washed once with PBS, then cell bodies were collected with a cell scraper in 1 mL PBS. Membranes were separated from their housing and placed into a lysis buffer from a Quick-RNA Microprep Kit (Zymo, R1050). Cell body and neurite samples from all wells of a 6-well plate were combined separately for each condition and used for RNA isolation. Following isolation, ribosomal RNA was depleted from 300 ng of the total RNA samples using the NEBNext rRNA Depletion Kit (NEB, E6310L) used for RNA-seq library prep with the NEBNext Ultra II Directional RNA Library Prep Kit (NEB, E7530L) and sequenced using an Illumina NextSeq 500.
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| Contributor(s) |
Wang E, Hildebrandt R |
| Citation(s) |
37296096 |
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| Submission date |
Jul 06, 2022 |
| Last update date |
Sep 08, 2023 |
| Contact name |
Eric T Wang |
| Organization name |
University of Florida
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| Street address |
2033 Mowry Rd
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| City |
Gainesville |
| State/province |
FL |
| ZIP/Postal code |
32610 |
| Country |
USA |
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| Platforms (1) |
| GPL19057 |
Illumina NextSeq 500 (Mus musculus) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA856295 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE207597_LR.xlsx |
23.2 Kb |
(ftp)(http) |
XLSX |
| GSE207597_TPM.xlsx |
2.5 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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