PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites

Danan, Charles H; Manickavel, Sudhir; Hafner, Markus

doi:10.1007/978-1-4939-3067-8_10

Cited by 65 publications

(39 citation statements)

References 38 publications

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“…PAR-CLIP PAR-CLIP was performed as described previously (Hafner et al 2010;Danan et al 2016). Briefly, for each PAR-CLIP experiment, 400 × 10 6 to 600 × 10 6 cells were cultured in medium containing 100 µM 4SU for 16 h. Cells were washed and resuspended in 15 mL of PBS and irradiated in 15-cm plates with 1.5 mJ/cm 2 312-nm wavelength UV.…”

Section: Recombinant Lin28 and Igf2bp3 Protein Expression And Purificmentioning

confidence: 99%

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3

Wang

Chim

et al. 2019

Genes Dev.

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Fetal hematopoietic stem and progenitor cells (HSPCs) hold promise to cure a wide array of hematological diseases, and we previously found a role for the RNA-binding protein (RBP) Lin28b in respecifying adult HSPCs to resemble their fetal counterparts. Here we show by single-cell RNA sequencing that Lin28b alone was insufficient for complete reprogramming of gene expression from the adult toward the fetal pattern. Using proteomics and in situ analyses, we found that Lin28b (and its closely related paralog, Lin28a) directly interacted with Igf2bp3, another RBP, and their enforced co-expression in adult HSPCs reactivated fetal-like B-cell development in vivo more efficiently than either factor alone. In B-cell progenitors, Lin28b and Igf2bp3 jointly stabilized thousands of mRNAs by binding at the same sites, including those of the B-cell regulators Pax5 and Arid3a as well as Igf2bp3 mRNA itself, forming an autoregulatory loop. Our results suggest that Lin28b and Igf2bp3 are at the center of a gene regulatory network that mediates the fetal-adult hematopoietic switch. A method to efficiently generate induced fetal-like hematopoietic stem cells (ifHSCs) will facilitate basic studies of their biology and possibly pave a path toward their clinical application.These authors contributed equally to this work Corresponding authors: stefan.muljo@nih.gov, markus.hafner@nih.gov Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/

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Section: Recombinant Lin28 and Igf2bp3 Protein Expression And Purificmentioning

confidence: 99%

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3

Wang

Chim

et al. 2019

Genes Dev.

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“…For each PAR-CLIP assay 1000 μl of Dynabeads (Invitrogen) and 800 μl of anti-EIF3B antibody (Bethyl A301-761A) were used. The remaining steps of the PAR-CLIP analysis was performed exactly as described in 5, 41 with the exception of using MNase at 5 U μl −1 for the on-bead digestion step.…”

Section: Methodsmentioning

confidence: 99%

A burst in T cell receptor translation mediated by eIF3 interactions with T cell receptor mRNAs

Silva

Ferguson

Smith

et al. 2019

Preprint

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39Activation of T cells requires a global surge in cellular protein synthesis, which is 40 accompanied by a large increase in translation initiation [1][2][3][4] . A central component of the 41 translation initiation machinery-the multi-subunit eukaryotic initiation factor 3 (eIF3)-is 42 rapidly turned on when quiescent T cells are stimulated 3 . However, the precise role eIF3 43 plays in activated T cells is not known. Using a global transcriptome cross-linking 44 approach, we show that human eIF3 interacts with a distinct set of mRNAs in activated 45 Jurkat cells. A subset of these mRNAs, including those encoding the T cell receptor (TCR) 46 subunits TCRA and TCRB, crosslink to eIF3 across the entire length of the mRNA. 47Main 55 Several lines of evidence indicate eIF3 serves specialized roles in cellular translation, by 56 recognizing specific RNA structures in the 5'-untranslated regions (5'-UTRs) of target 57 mRNAs 5 , binding the 7-methyl-guanosine (m 7 G) cap 6 or through interactions with N-6-58 methyl-adenosine (m 6 A) post-transcriptional modifications in mRNAs 7 . Binding to these 59 cis-regulatory elements in mRNA can lead to translation activation or repression, 60 depending on the RNA sequence and structural context 5,7,8 . These functions for eIF3 can 61 aid cell proliferation 5 , or allow cells to rapidly adapt to stress such as heat shock 7 . 62 Additionally, eIF3 plays an important role in the development of specific tissues 9-11 . 63 64 In the immune system, T cell activation involves a burst in translation 1-4 and correlates 65 with assembly of subunit EIF3J with the main eIF3 complex 3 . However, whether eIF3 66 serves a general or more specific role in T cell activation is not known. To delineate how 67 eIF3 contributes to T cell activation, we first identified mRNAs that directly interact with 68 eIF3 in Jurkat cells activated for 5 hours with ionomycin and phorbol 12-myristate 13-69 acetate (I+PMA), or in non-activated Jurkat cells as a control, using photoactivatable 70 ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) 5,12,13 (Fig. 71 1a). We used Jurkat cells as a model for T cells, as PAR-CLIP experiments require a 72 large number of cells labeled with 4-thiouridine at a non-toxic concentration 14 . Jurkat cells 73 also have a defined T cell receptor and transcriptome, avoiding the donor-to-donor 74 variability of primary T cells. In the Jurkat PAR-CLIP experiments, RNA crosslinked to 75 eight of the thirteen eIF3 subunits, as identified by mass spectrometry: subunits EIF3A, 76 EIF3B, EIF3D and EIF3G as seen in HEK293T cells 5 , as well as subunits EIF3C, EIF3E, 77 De Silva et al. 4EIF3F, and EIF3L (Fig. 1b, Extended Data Fig. 1, Supplementary Table 1). In activated 78 Jurkat cells, eIF3 crosslinked to a substantially larger number of mRNAs compared to the 79 non-activated cells (Extended Data Figs. 2 and 3, Supplementary Tables 2 and 3). 80 Notably, eIF3 interacted with a completely new suite of mRNAs in activated Jurkat cells, 81 co...

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“…A mass spectrometry study has compared this protocol to UV-C crosslinking to conclude that the efficiency of the two approaches is quite similar across all RBPs (Castello et al, 2012). The UV-A crosslinking is limited to biological systems where the photoactivatable nucleosides can be efficiently incorporated, and it recommends use of >100 million cells (Danan et al, 2016), while 1 million or less can be used by standard variants of CLIP. Moreover, prolonged preincubation with 6SG or 4SU can cause cellular toxicity, including stress responses and inhibition of rRNA synthesis (Burger et al, 2013; Huppertz et al, 2014) and therefore care needs to be taken to monitor the cellular response to these ribonucleosides (Burger et al, 2013; Huppertz et al, 2014).…”

Section: The Clip Methodsmentioning

confidence: 99%

The Future of Cross-Linking and Immunoprecipitation (CLIP)

Ule

Hwang

Darnell

2018

Cold Spring Harb Perspect Biol

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To understand the assembly and functional outcomes of protein-RNA regulation, it is crucial to precisely identify the positions of such interactions. Crosslinking and immunoprecipitation (CLIP) serves this purpose by exploiting covalent protein-RNA crosslinking and RNA fragmentation, along with a series of stringent purification and quality control steps to prepare cDNA libraries for sequencing. Here we describe the core steps of CLIP, its primary variations and the approaches to data analysis. We present the application of CLIP to studies of specific cell types in genetically engineered mice, and discuss the mechanistic and physiologic insights that have already been gained from studies employing CLIP. We conclude by discussing the future opportunities for CLIP, including studies of human postmortem tissues from disease patients and controls, RNA epigenetic modifications, and RNA structure. These and other applications of CLIP will continue to unravel fundamental gene regulatory mechanisms, while providing important biologic and clinically relevant insights.

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PAR-CLIP: A Method for Transcriptome-Wide Identification of RNA Binding Protein Interaction Sites

Cited by 65 publications

References 38 publications

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3

Enhancement of LIN28B-induced hematopoietic reprogramming by IGF2BP3

A burst in T cell receptor translation mediated by eIF3 interactions with T cell receptor mRNAs

The Future of Cross-Linking and Immunoprecipitation (CLIP)

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