Quantitative Profiling of In Vivo-assembled RNA-Protein Complexes Using a Novel Integrated Proteomic Approach*

Tsai, Becky Pinjou; Wang, Xiaorong; Huang, Lan; Waterman, Marian L.

doi:10.1074/mcp.a110.007385

Cited by 40 publications

(54 citation statements)

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“…The in vitro approaches generally employ a synthetic RNA bait to capture and identify proteins from cellular extracts [43,88,90]. In contrast, the in vivo approaches capture the RNA-protein complexes present in the cell [45,46,85,92]. While the in vivo methods preserve the context of true RNA-protein interactions, they are more technically challenging, especially if the target RNA is of low abundance in the cell.…”

Section: Rna-centric Methodsmentioning

confidence: 99%

“…Studies using in vitro approaches or performing purifications in native conditions have generally found association between the RNA of interest and highly abundant proteins in the cell, such as hnRNPs [85,91,92]. Whether these represent real biological interactions or non-specific associations is unclear because only a handful of RNAs have been purified to date.…”

Section: Rna-centric Methodsmentioning

confidence: 99%

“…Whether these represent real biological interactions or non-specific associations is unclear because only a handful of RNAs have been purified to date. To address this, a recent study made use of UV crosslinking and purified RNA complexes under fully denaturing conditions (using 8 M urea), which will only capture in vivo crosslinked complexes [85]. Using this approach, there were clear differences in the proteins identified after purifications performed in native and denaturing conditions.…”

Section: Rna-centric Methodsmentioning

confidence: 99%

“…Similar to the protein-centric methods, great care must be taken to select informative negative controls for the RNA-centric methods. Controls that are often used include a different cellular RNA [92], sequences lacking known protein-binding structures [85-91], tag-only controls [44], antisense RNA [71,98] or non-specific RNA sequences [99]. In these cases, any non-specific protein interactions due to abundance, nucleic acid binding or the tag itself should be identical for the target RNA and controls.…”

Section: Rna-centric Methodsmentioning

confidence: 99%

“…Unlike sequencing methods that enable nucleic acid amplification, the amount of protein purified in these experiments cannot be amplified. For this reason, RNA-centric methods have mostly been applied to highly abundant RNAs, such as 7SK [44], snRNPs [100], Let-7 [99] and IRES [85]. More recently, these approaches have been used to define proteins associated with all mRNA by UV crosslinking RNA-protein complexes, capturing polyadenylated transcripts using oligo-dT coupled magnetic beads, and detecting associated proteins by quantitative MS [45,46,94].…”

Section: Rna-centric Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Methods for comprehensive experimental identification of RNA-protein interactions

2014

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Section: Rna-centric Methodsmentioning

confidence: 99%

Section: Rna-centric Methodsmentioning

confidence: 99%

Section: Rna-centric Methodsmentioning

confidence: 99%

Section: Rna-centric Methodsmentioning

confidence: 99%

Section: Rna-centric Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Methods for comprehensive experimental identification of RNA-protein interactions

2014

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Adaptation of Tri‐molecular fluorescence complementation allows assaying of regulatory Csr RNA‐protein interactions in bacteria

Gelderman

Sivakumar

Lipp

et al. 2014

Biotech & Bioengineering

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sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo.

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Nanog RNA‐binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells

Guo

Xue

Yang

et al. 2016

Cell Biology International

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Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs.

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Quantitative Profiling of In Vivo-assembled RNA-Protein Complexes Using a Novel Integrated Proteomic Approach*

Cited by 40 publications

References 0 publications

Methods for comprehensive experimental identification of RNA-protein interactions

Methods for comprehensive experimental identification of RNA-protein interactions

Adaptation of Tri‐molecular fluorescence complementation allows assaying of regulatory Csr RNA‐protein interactions in bacteria

Nanog RNA‐binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells

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