Modeling Hypoxia-Induced Neuropathies Using a Fast and Scalable Human Motor Neuron Differentiation System

Hudish, Laura I.; Bubak, Andrew N.; Triolo, Taylor M.; Niemeyer, Christy S.; Lorberbaum, David S.; Sussel, Lori; Nagel, Maria A.; Taliaferro, J. Matthew; Russ, Holger A.

doi:10.1016/j.stemcr.2020.04.003

Cited by 11 publications

(20 citation statements)

References 37 publications

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“…We found that the 3′ UTRs of the chosen neurite-localized genes were more conserved than other 3′ UTRs, suggesting that they may contain functional localization elements (Figure 1C). Using RNAseq data from fractionated human motor neurons (Goering et al, 2020a;Hudish et al, 2020), we found that the localization of the human orthologs of these transcripts was very similar to their mouse counterparts, further suggesting that conserved elements within these transcripts mediate their transport (Figure S1B).…”

Section: Identification Of 3′ Utrs Sufficient For Rna Localization Activitymentioning

confidence: 88%

“…In order to study RNA localization in neuronal cells on a transcriptomic scale, we have developed and used a mechanical fractionation technique that makes use of cell growth on microporous membranes (Arora et al, 2021;Goering et al, 2020a;Hudish et al, 2020;Taliaferro et al, 2016) (Figure 1A). With this technique, cells are plated on the top of membranes whose pores allow neurite growth to the underside of the membrane.…”

Section: Identification Of 3′ Utrs Sufficient For Rna Localization Activitymentioning

confidence: 99%

“…Reporter and control iPS cells were then differentiated into motor neurons and mechanically fractionated into cell body and neurite fractions as described previously (Hudish et al, 2020). (Figure 1A, 5F).…”

Section: Peak Oligonucleotides Are Sufficient To Drive Rna Localization In Human Motor Neuronsmentioning

confidence: 99%

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High-throughput identification of RNA localization elements reveals a regulatory role for A/G rich sequences

Arora

Gutierrez

Eletto

et al. 2021

Preprint

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Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements within them that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we designed and deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3′ UTRs. We identified peaks of regulatory activity within several 3′ UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse cells as well as active in human neurons. The active localization elements were enriched in adenosine and guanosine residues and were tens to hundreds of nucleotides long as shortening of the elements led to significantly reduced activity. Using RNA affinity and mass spectrometry, we found that the RNA-binding protein Unk was associated with the active localization elements. Depletion of Unk in cells reduced the neurite- enrichment of the elements, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization.

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Section: Identification Of 3′ Utrs Sufficient For Rna Localization Activitymentioning

confidence: 88%

Section: Identification Of 3′ Utrs Sufficient For Rna Localization Activitymentioning

confidence: 99%

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High-throughput identification of RNA localization elements reveals a regulatory role for A/G rich sequences

Arora

Gutierrez

Eletto

et al. 2021

Preprint

Self Cite

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“…Dissociated Accutase passaged single neurons d11 (400K) were plated in motor neuron differentiation media in 6 well hanging inserts with Boyden chamber device (1 μm pore, Falcon Cell Culture Inserts, #10289270, Thermo) for axon isolation (d22) similar to recent protocols [4,18]. Transmembrane filters were coated on both sides with poly-d-lysine and only on bottom side with laminin.…”

Section: Axon Isolationmentioning

confidence: 99%

Peripheral neuropathy linked mRNA export factor GANP reshapes gene regulation in human motor neurons

Woldegebriel

Kvist

White

et al. 2021

Preprint

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Loss-of-function of the mRNA export protein GANP (MCM3AP gene) cause early-onset sensorimotor neuropathy, characterised by axonal degeneration in long peripheral nerves. GANP functions as a scaffold at nuclear pore complexes, contributing to selective nuclear export of mRNAs. Here, we aimed to identify motor neuron specific transcripts that are regulated by GANP and may be limiting for local protein synthesis in motor neuron axons. We compared motor neurons with a gene edited mutation in the Sac3 mRNA binding domain of GANP to isogenic controls. We also examined patient-derived motor neurons. RNA sequencing of motor neurons as well as nuclear and axonal subcompartments showed that mutant GANP had a profound effect on motor neuron transcriptomes, with alterations in nearly 40 percent of all expressed genes and broad changes in splicing. Expression changes in multiple genes critical for neuronal functions, combined with compensatory upregulation of protein synthesis and early-stage metabolic stress genes, indicated that RNA metabolism was abnormal in GANP-deficient motor neurons. Surprisingly, limited evidence was found for large-scale nuclear retention of mRNA. This first study of neuropathy-linked GANP defects in human motor neurons shows that GANP has a wide gene regulatory role in a disease-relevant cell type that requires long-distance mRNA transport.

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“…However, it has been unclear how widespread this effect is and whether it was driven primarily by tandem UTRs or ALEs. To address this, we used LABRAT to analyze the relative APA status of 26 paired transcriptomic datasets from cell body and projection samples from neuronal cells, NIH 3T3 cells, and MDA-MB231 cells (Farris et al, 2019;Goering et al, 2019;Hudish et al, 2020;Mardakheh et al, 2015;Minis et al, 2013;Taliaferro et al, 2016;Tushev et al, 2018;Wang et al, 2017;Zappulo et al, 2017).…”

Section: Alternative Polyadenylation Isoforms Are Differentially Locamentioning

confidence: 99%

LABRAT reveals association of alternative polyadenylation with transcript localization, RNA binding protein expression, transcription speed, and cancer survival

Goering

Engel

Gillen

et al. 2020

Preprint

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The sequence content of the 3′ UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging. To combat this problem, we developed LABRAT, an APA quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between APA and expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated.

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Modeling Hypoxia-Induced Neuropathies Using a Fast and Scalable Human Motor Neuron Differentiation System

Cited by 11 publications

References 37 publications

High-throughput identification of RNA localization elements reveals a regulatory role for A/G rich sequences

High-throughput identification of RNA localization elements reveals a regulatory role for A/G rich sequences

Peripheral neuropathy linked mRNA export factor GANP reshapes gene regulation in human motor neurons

LABRAT reveals association of alternative polyadenylation with transcript localization, RNA binding protein expression, transcription speed, and cancer survival

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