Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis

Han, Hong; Best, Andrew; Braunschweig, Ulrich; Mikolajewicz, Nicholas; Li, Jack Daiyang; Roth, Jonathan; Chowdhury, Fuad; Mantica, Federica; Nabeel‐Shah, Syed; Parada, Guillermo E.; Brown, Kevin R.; O’Hanlon, Dave; Wei, Jiarun; Yao, Yuxi; Zid, Abdelrahman Abou; Comsa, Lim Caden; Jen, Mark; Wang, Jenny; Datti, Alessandro; Gonatopoulos-Pournatzis, Thomas; Weatheritt, Robert J.; Greenblatt, Jack; Wrana, Jeffrey L.; Irimia, Manuel; Gingras, Anne‐Claude; Moffat, Jason; Blencowe, Benjamin J.

doi:10.1016/j.molcel.2022.06.036

Cited by 14 publications

(12 citation statements)

References 103 publications

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“…Interestingly, however, the published single-cell RNA-sequencing (RNA-seq) data on Ptbp1 KO retina appear to show elevated rod photoreceptor cells, similar to the original report by Zhang and colleagues (Fu et al 2020), but those rod cells could not be traced to Müller glia (Hoang et al 2022). The authors claimed to have detected little changes in gene expression in homozygous Ptbp1 KO Müller glia (Hoang et al 2022), which is surprising because Ptbp1 KD has been shown to induce widespread changes in gene expression at both transcriptional and posttranscriptional levels (Han et al 2022;Xue et al 2009Xue et al , 2013Xue et al , 2016. One should consider overcompensation by Ptbp2 in Ptbp1 KO cells as a possibility (see below).…”

Section: Failed Atn Conversion Based On Lineage Tracing Of Astrocytesmentioning

confidence: 53%

Therapeutic Potential of PTB Inhibition Through Converting Glial Cells to Neurons in the Brain

Mobley

2023

Annu. Rev. Neurosci.

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Cell replacement therapy represents a promising approach for treating neurodegenerative diseases. Contrary to the common addition strategy to generate new neurons from glia by overexpressing a lineage-specific transcription factor(s), a recent study introduced a subtraction strategy by depleting a single RNA-binding protein, Ptbp1, to convert astroglia to neurons not only in vitro but also in the brain. Given its simplicity, multiple groups have attempted to validate and extend this attractive approach but have met with difficulty in lineage tracing newly induced neurons from mature astrocytes, raising the possibility of neuronal leakage as an alternative explanation for apparent astrocyte-to-neuron conversion. This review focuses on the debate over this critical issue. Importantly, multiple lines of evidence suggest that Ptbp1 depletion can convert a selective subpopulation of glial cells into neurons and, via this and other mechanisms, reverse deficits in a Parkinson's disease model, emphasizing the importance of future efforts in exploring this therapeutic strategy.

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Section: Failed Atn Conversion Based On Lineage Tracing Of Astrocytesmentioning

confidence: 53%

Therapeutic Potential of PTB Inhibition Through Converting Glial Cells to Neurons in the Brain

Mobley

2023

Annu. Rev. Neurosci.

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“…Prior to converting RNA samples into cDNA, input RNA must undergo L3 adapter ligation. Of note, for certain spliceosome subunits (e.g., U2AF65 and PUF60), L3 adapter ligation to the immunoprecipitated protein-RNA complexes does not occur efficiently, presumably due to steric hinderance ( Shao et al, 2014 ; Han et al, 2022 ). In such cases, we recommend that the IP samples should be processed along with inputs so that, after immunoprecipitation and three high-salt buffer washes (Step B12), you proceed directly to radiolabeling (Step C11) and omit the dephosphorylation and adapter ligation steps described above in section C. Isolate the RNA from the membrane as detailed above and continue with the steps noted below for input samples.…”

Section: Methodsmentioning

confidence: 99%

“…Our data yielded high-resolution RNA-binding maps for SP1, as well as the cleavage and polyadenylation complex subunits, and unraveled a previously unknown function of SP1 in alternative cleavage and polyadenylation regulation. We have used this protocol with several cell lines (e.g., mouse neuroblastoma N2a cells, cgr8 embryonic stem cells, HeLa cells, glioblastoma, and HEK293 cells) to identify RNA targets of RBPs involved in diverse functions, including splicing regulation, mRNA stability, mRNA export, 5′ capping, and mRNA modifications ( Nitoiu et al, 2021; Han et al, 2017 and 2022; Nabeel-Shah et al, 2022 ). Our revised iCLIP protocol combines the improvements previously implemented in eCLIP (e.g., enhanced adapter ligation and SMI), with the optimization of several steps in the original iCLIP method, including improved circularization of the cDNA by increasing the incubation time and adding Betain in the reaction mixture, which helps the circularization of difficult-to-ligate substrates.…”

Section: Introductionmentioning

confidence: 99%

Revised iCLIP-seq Protocol for Profiling RNA–protein Interaction Sites at Individual Nucleotide Resolution in Living Cells

Nabeel‐Shah¹,

Greenblatt²

2023

BIO-PROTOCOL

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Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins’ (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview

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“…Interestingly, both PRMT5 and HTT are essential for neuronal stem cell proliferation and self-renewal 25,30,3113,18,19 . Since splicing and RNA metabolism have been shown to play a central role in neurogenesis, neuronal differentiation, and neurodegenerative diseases [33][34][35][36][37][38] it is plausible that PRMT5 may regulate these processes via RNA splicing, and that this role may intersect with HTT.…”

Section: Introductionmentioning

confidence: 99%

PRMT5 is required for full-lengthHTTexpression by repressing multiple proximal intronic polyadenylation sites

AlQazzaz,

Ciamponi,

et al. 2024

Preprint

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Expansion of the CAG trinucleotide repeat tract in exon 1 of theHuntingtin(HTT) gene above a threshold of ∼36 repeats causes Huntington’s disease (HD) through the expression of a polyglutamine-expanded form of the HTT protein. This mutation triggers wide-ranging cellular and biochemical pathologies leading to cognitive, motor, and psychiatric symptoms in HD patients. As accurate splicing is required to produce the full-length HTT protein of ∼348 kDa, targetingHTTsplicing with small molecule drugs is a compelling approach to lower HTT protein levels to treat HD, and splice modulators are being tested in the clinic. Here, we identify PRMT5 as a novel regulator ofHTTmRNA splicing and alternative polyadenylation. PRMT5 inhibition disrupts the splicing ofHTTintrons 9 and 10, leading to activation of multiple proximal intronic polyadenylation sites within these introns and promoting premature termination, cleavage and polyadenylation (PCPA) of theHTTmRNA, thus lowering total HTT protein levels. We also detected increasing levels of these truncated, intron-containingHTTtranscripts across a series of neuronal differentiation samples which correlated with lower PRMT5 expression. Notably, PRMT5 inhibition in glioblastoma (GBM) stem cells potently induced neuronal differentiation. We posit that PRMT5-mediated regulation of intronic polyadenylation, premature termination and cleavage of theHTTmRNA modulates HTT expression and plays an important role during embryonic development and neuronal differentiation.

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Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis

Cited by 14 publications

References 103 publications

Therapeutic Potential of PTB Inhibition Through Converting Glial Cells to Neurons in the Brain

Therapeutic Potential of PTB Inhibition Through Converting Glial Cells to Neurons in the Brain

Revised iCLIP-seq Protocol for Profiling RNA–protein Interaction Sites at Individual Nucleotide Resolution in Living Cells

PRMT5 is required for full-lengthHTTexpression by repressing multiple proximal intronic polyadenylation sites

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