Genome-wide analysis of RNA and protein localization and local translation in mESC-derived neurons

Ludwik, Katarzyna A.; Kuegelgen, Nicolai von; Chekulaeva, Marina

doi:10.1016/j.ymeth.2019.02.002

Cited by 14 publications

(8 citation statements)

References 33 publications

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“…RNA samples collected from soma and neurites were then analyzed by quantitative RT-PCR. We observed enrichment of the well-characterized neuronal projections markers TAGLN, COL3A1 and MAPKAK2 in the neurite compartment, and enrichment of GNG3 and ENO2 in the soma ( Figure 1E ), consistent with the known localization of these transcripts in neurons (Ludwik et al, 2019).…”

Section: Resultssupporting

confidence: 80%

Digital color-coded molecular barcoding reveals dysregulation of common FUS and FMRP targets in soma and neurites of ALS mutant motoneurons

Garone

Salerno

Rosa

2022

Preprint

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Mutations in RNA binding proteins (RBPs) have been linked to the motor neuron disease amyotrophic lateral sclerosis (ALS). Extensive auto-regulation, cross-regulation, cooperation and competition mechanisms among RBPs are in place to ensure proper expression levels of common targets, often including other RBPs and their own transcripts. Moreover, several RBPs play a crucial role in the nervous system by localizing target RNAs in specific neuronal compartments. These include the RBPs FUS, FMRP and HuD. ALS mutations in a given RBP are predicted to produce a broad impact on such delicate equilibrium. Here we studied the effects of the severe FUS-P525L mutation on common FUS and FMRP targets. Expression profiling by digital color-coded molecular barcoding in cell bodies and neurites of human iPSC-derived motor neurons revealed altered levels of transcripts involved in cytoskeleton, neural projection and synapses. One of the common targets is HuD, which is upregulated because of the loss of FMRP binding to its 3’UTR due to mutant FUS competition. Notably, many genes are commonly altered upon FUS mutation or HuD overexpression, suggesting that a substantial part of the effects of mutant FUS on the motor neuron transcriptome could be due to HuD gain-of-function. Among altered transcripts we also identified other common FUS and FMRP targets, namely MAP1B, PTEN, and AP2B1, that are upregulated upon loss of FMRP binding on their 3’UTR in FUS-P525L motor neurons. This work demonstrates that the impairment of FMRP function by mutant FUS might alter the expression of several genes, including new possible biomarkers and therapeutic targets for ALS.

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Section: Resultssupporting

confidence: 80%

Digital color-coded molecular barcoding reveals dysregulation of common FUS and FMRP targets in soma and neurites of ALS mutant motoneurons

Garone

Salerno

Rosa

2022

Preprint

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“…Similar to microdissection, this approach produces a heterogeneous population of neuronal types and can be combined with FACS (Poulopoulos et al, 2019). Subcellular compartments of neurons can also be separated by culturing the cells (c) in compartmentalized microfluidic chambers (Briese et al, 2016;Taylor, Dieterich, Ito, Kim, & Schuman, 2010) or (d) on microporous membranes (Ciolli Mattioli et al, 2019;Ludwik, von Kuegelgen, & Chekulaeva, 2019;Pertz, Hodgson, Klemke, & Hahn, 2006;Taliaferro et al, 2016;Zappulo et al, 2017) The latter separate cell bodies, which grow on top of the membrane, from neurites, which stretch through the pores and emerge on the lower side of the membrane. This method can be easily scaled up to produce enough material for multiple omics analyses, but does not always permit a separation of axons from dendrites.…”

Section: Isolation Of Subcellular Neuronal Compartments For Rna Sequencingmentioning

confidence: 99%

Conservation of a core neurite transcriptome across neuronal types and species

Kügelgen

Chekulaeva

2020

WIREs RNA

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“…For quantifying the abundance of library sequences in neurite and soma, CAD and Neuro-2a cells were grown on Millicell Hanging Cell Culture Inserts with a pore size of 3 μm (Millipore #MCSP06H48), adapting experimental pipelines described earlier (Ludwik et al, 2019;Taliaferro et al, 2016;Zappulo et al, 2017). Prior to seeding the cells, the bottom of the insert was coated with 100 ul Matrigel (Corning #356237, diluted to 3 mg/ml with PBS).…”

Section: Library Transfection and Neurite-and Soma-specific Rna Extractionmentioning

confidence: 99%

A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons

Mikl

Eletto

Lee

et al. 2021

Preprint

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Asymmetric subcellular localization of mRNA is a common cellular phenomenon that is thought to contribute to spatial gene regulation. In highly polar neurons, subcellular transcript localization and translation are thought to enhance cellular efficiency and timely responses to external cues. Although mRNA localization has been observed in many tissues and numerous examples of the functional importance of this process exist, we still lack a systematic understanding of how the transcript sorting machinery works in a sequence-specific manner. Here, we addressed these gaps by combining subcellular transcriptomics and rationally designed sequence libraries. We developed a massively parallel reporter assay (MPRA) for mRNA localization and tested ~50,000 sequences for their ability to drive RNA localization to neurites of neuronal cell lines. By scanning the 3'UTR of >300 genes we identified many previously unknown localization regions and mapped the localization potential of endogenous sequences. Our data suggest two ways the localization potential can be encoded in the 3'UTR: focused localization motifs and broadly encoded localization potential based on small contributions. We identified sequence motifs enriched in dendritically localized transcripts and tested the potential of these motifs to affect the localization behavior of an mRNA. This assay revealed sequence elements with the ability to bias localization towards neurite as well as soma. Depletion of RNA binding proteins predicted or experimentally shown to bind these motifs abolished the effect on localization, suggesting that these motifs act by recruiting specific RNA-binding proteins. Based on our dataset we developed machine learning models that accurately predict the localization behavior of novel sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native transcripts. Applying similar systematic high-throughput approaches to other cell types will open the door for a comparative perspective on RNA localization across tissues and reveal the commonalities and differences of this crucial regulatory mechanism.

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Genome-wide analysis of RNA and protein localization and local translation in mESC-derived neurons

Cited by 14 publications

References 33 publications

Digital color-coded molecular barcoding reveals dysregulation of common FUS and FMRP targets in soma and neurites of ALS mutant motoneurons

Digital color-coded molecular barcoding reveals dysregulation of common FUS and FMRP targets in soma and neurites of ALS mutant motoneurons

Conservation of a core neurite transcriptome across neuronal types and species

A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons

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