Affinity Purification of Long Noncoding RNA–Protein Complexes from Formaldehyde Cross-Linked Mammalian Cells

2014 ◽  
pp. 81-86 ◽  
Author(s):  
Chenguang Gong ◽  
Lynne E. Maquat
Keyword(s):  
Long Noncoding Rna ◽  
Mammalian Cells ◽  
Noncoding Rna ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Rna Protein Complexes

scholarly journals Chromosome-associated RNA–protein complexes promote pairing of homologous chromosomes during meiosis in Schizosaccharomyces pombe

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Da-Qiao Ding ◽  
Kasumi Okamasa ◽  
Yuki Katou ◽  
Eriko Oya ◽  
Jun-ichi Nakayama ◽  
...  
Keyword(s):  
Phase Separation ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Homologous Pairing ◽  
Homologous Chromosomes ◽  
Rna Protein Complexes ◽  
Chromosomal Loci

AbstractPairing of homologous chromosomes in meiosis is essential for sexual reproduction. We have previously demonstrated that the fission yeast sme2 RNA, a meiosis-specific long noncoding RNA (lncRNA), accumulates at the sme2 chromosomal loci and mediates their robust pairing in meiosis. However, the mechanisms underlying lncRNA-mediated homologous pairing have remained elusive. In this study, we identify conserved RNA-binding proteins that are required for robust pairing of homologous chromosomes. These proteins accumulate mainly at the sme2 and two other chromosomal loci together with meiosis-specific lncRNAs transcribed from these loci. Remarkably, the chromosomal accumulation of these lncRNA–protein complexes is required for robust pairing. Moreover, the lncRNA–protein complexes exhibit phase separation properties, since 1,6-hexanediol treatment reversibly disassembled these complexes and disrupted the pairing of associated loci. We propose that lncRNA–protein complexes assembled at specific chromosomal loci mediate recognition and subsequent pairing of homologous chromosomes.

scholarly journals Isolation of Endogenously Assembled RNA-Protein Complexes Using Affinity Purification Based on Streptavidin Aptamer S1

2015 ◽  
Vol 16 (9) ◽  
pp. 22456-22472 ◽  
Author(s):  
Yangchao Dong ◽  
Jing Yang ◽  
Wei Ye ◽  
Yuan Wang ◽  
Chuantao Ye ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Streptavidin Aptamer ◽  
Rna Protein Complexes

scholarly journals Enzymatic RNA Biotinylation for Affinity Purification and Identification of RNA-protein Interactions

2020 ◽  
Author(s):  
Kayla N. Busby ◽  
Amitkumar Fulzele ◽  
Dongyang Zhang ◽  
Eric J. Bennett ◽  
Neal K. Devaraj
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Noncoding Rna ◽  
Affinity Purification ◽  
Functional Characterization ◽  
Selective Enrichment ◽  
Binding Partners ◽  
Nucleotide Mutation ◽  
Rna Biology ◽  
And Function

ABSTRACTThroughout their cellular lifetime, RNA transcripts are bound to proteins, playing crucial roles in RNA metabolism, trafficking, and function. Despite the importance of these interactions, identifying the proteins that interact with an RNA of interest in mammalian cells represents a major challenge in RNA biology. Leveraging the ability to site-specifically and covalently label an RNA of interest using E. Coli tRNA guanine transglycosylase and an unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology.

scholarly journals Identification of heat-inducible long noncoding RNA MALAT1-containing novel nuclear (HiNoCo) body

2021 ◽  
Author(s):  
Rena Onoguchi-Mizutani ◽  
Yoshitaka Kirikae ◽  
Yoko Ogura ◽  
Tony Gutschner ◽  
Sven Diederichs ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Response ◽  
Long Noncoding Rna ◽  
Mammalian Cells ◽  
Noncoding Rna ◽  
Heat Shock Factor ◽  
Nuclear Bodies ◽  
Cajal Body ◽  
Heat Shock Factor 1 ◽  
Shock Response

The heat shock response is critical for the survival of all organisms. Metastasis-associated long adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA localized in nuclear speckles, but its physiological role remains elusive. Here, we show that heat shock induces translocation of MALAT1 to a distinct nuclear body named heat shock-inducible noncoding RNA-containing nuclear (HiNoCo) body in mammalian cells. The MALAT1 knockout A549 cells showed reduced proliferation after heat shock. The HiNoCo body, formed by a nearby nuclear speckle, is distinct from any other known nuclear bodies, including the nuclear stress body, Cajal body, germs, paraspeckles, nucleoli, and promyelocytic leukemia body. The formation of HiNoCo body is reversible and independent of heat shock factor 1, the master transcription regulator of the heat shock response. Our results suggest the HiNoCo body participates in heat shock factor 1-independent heat shock responses in mammalian cells.

scholarly journals Long noncoding RNA and protein abundance in lncRNPs

RNA ◽  
2021 ◽  
pp. rna.078971.121
Author(s):  
Man Wu ◽  
Liang-Zhong Yang ◽  
Ling-Ling Chen
Keyword(s):  
Gene Regulation ◽  
Protein Expression ◽  
Relative Abundance ◽  
Long Noncoding Rna ◽  
Noncoding Rna ◽  
Protein Complexes ◽  
Noncoding Rnas ◽  
Protein Abundance ◽  
Interacting Proteins ◽  
Low Levels

Although long noncoding RNAs (lncRNAs) are generally expressed at low levels, emerging evidence has revealed that many play important roles in gene regulation by a variety of mechanisms as they engage with proteins. Given that the abundance of proteins often greatly exceeds that of their interacting lncRNAs, quantification of the relative abundance, or even the exact stoichiometry in some cases, within lncRNA-protein complexes is helpful for understanding of the mechanism(s) of action of lncRNAs. We discuss methods used to examine lncRNA and protein expression at the single cell, sub-cellular and sub-organelle levels, the average and local lncRNA concentration in cells, as well as how lncRNAs can modulate the functions of their interacting proteins even at a low stoichiometric concentration.

scholarly journals Tandem Affinity Purification of Protein Complexes from Mammalian Cells

BioTechniques ◽  
2002 ◽  
Vol 33 (2) ◽  
pp. 267-270 ◽  
Author(s):  
D.M. Cox ◽  
M. Du ◽  
X. Guo ◽  
K.W.M. Siu ◽  
J.C. McDermott
Keyword(s):  
Mammalian Cells ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Tandem Affinity Purification

scholarly journals RNA Proximity Labeling: A New Detection Tool for RNA–Protein Interactions

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2270
Author(s):  
Ronja Weissinger ◽  
Lisa Heinold ◽  
Saira Akram ◽  
Ralf-Peter Jansen ◽  
Orit Hermesh
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Cellular Functions ◽  
Cellular Compartments ◽  
Rna Protein Complexes

Multiple cellular functions are controlled by the interaction of RNAs and proteins. Together with the RNAs they control, RNA interacting proteins form RNA protein complexes, which are considered to serve as the true regulatory units for post-transcriptional gene expression. To understand how RNAs are modified, transported, and regulated therefore requires specific knowledge of their interaction partners. To this end, multiple techniques have been developed to characterize the interaction between RNAs and proteins. In this review, we briefly summarize the common methods to study RNA–protein interaction including crosslinking and immunoprecipitation (CLIP), and aptamer- or antisense oligonucleotide-based RNA affinity purification. Following this, we focus on in vivo proximity labeling to study RNA–protein interactions. In proximity labeling, a labeling enzyme like ascorbate peroxidase or biotin ligase is targeted to specific RNAs, RNA-binding proteins, or even cellular compartments and uses biotin to label the proteins and RNAs in its vicinity. The tagged molecules are then enriched and analyzed by mass spectrometry or RNA-Seq. We highlight the latest studies that exemplify the strength of this approach for the characterization of RNA protein complexes and distribution of RNAs in vivo.

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