We thank Eric L Van Nostrand and Gene W Yeo for helpful guidance with the original protocol. K.Z. was supported by National Key R&#38;D Program of China (2016YFA0500902, 2018YFC1003500), and National Natural Science Foundation of China (31771653). L.Y. was supported by National Natural Science Foundation of China (81471502, 31871503) and Innovative and Entrepreneurial Program of Jiangsu Province.

<ol>
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The authors have nothing to disclose.

With increasing understanding of the universal role of RBPs under both biological and pathological contexts, the CLIP methods have been widely utilized to reveal the molecular function of RBPs20,32,33,34,35. The protocol described here represents an adapted application of the eCLIP method to mouse testis.
One challenge in performing...

Mammalian testis represents an excellent developmental model wherein an intricate cell differentiation program runs cyclically to yield numerous spermatozoa. An unique value of this model lies in the emergence of transcriptional inactivation at certain stages of spermatogenesis, typically when meiotic sex chromosome inactivation (MSCI) occurs1,2 and when round spermatids undergo drastic nuclear compaction during spermiogenesis3. These inconsecutive transcriptional events necessitate post-transcriptional gene regulation, in which RNA-binding proteins (RBPs) play a crucial role, shaping transcriptome and maintaining male fertility.
To identify the bona fide RNA targets of an individual RBP in vivo, the crosslinking immunoprecipitation (CLIP) method was developed4,5, based on but beyond the regular RNA immunoprecipitation (RIP)6,7, by incorporation of key steps including ultraviolet (UV) crosslinking, stringent wash and gel transfer to improve signal specificity. The advanced application of the CLIP combined with high-throughput sequencing has provoked large interest in profiling protein-RNA interaction at genome-wide levels8. In addition to genetic studies on RBP function, such biochemical methods that identify the direct interplay of endogenous protein and RNA have been indispensable to accurately elucidate the RNA regulatory roles of RBPs. For example, MOV10L1 is a testis-specific RNA helicase required for male fertility and the Piwi-interacting RNA (piRNA) biogenesis9. Its paralogue MOV10 is known as a ubiquitously expressed and multifunctional RNA helicase with roles in multiple aspects of RNA biology10,11,12,13,14,15,16,17,18. By employing the conventional CLIP-seq, we found that MOV10L1 binds and regulates primary piRNA precursors to initiate early piRNA processing19,20, and that MOV10 binds mRNA 3&#39; UTRs and as well as noncoding RNA species in testicular germ cells (data not shown).
Nevertheless, CLIP is originally a laborious, radioactive procedure followed by sequencing library preparation with a remarkable loss of CLIP tags. In the conventional CLIP, a cDNA library is prepared using adapters ligated at both RNA extremities. After protein digestion, crosslinked short polypeptides remain attached to RNA fragments. This crosslinking mark partially blocks reverse transcriptase (RTase) progression during cDNA synthesis, resulting in truncated cDNAs which represent about 80% of the cDNA library21,22. Thus, only cDNA fragments resulting from RTase bypassing the crosslinking site (read-through) are sequenced. Recently, various CLIP approaches, such as PAR-CLIP, iCLIP, eCLIP and uvCLAP, have been employed to identify crosslink sites of RBPs in living cells. PAR-CLIP involves the application of 365 nm UV radiation and photoactivatable nucleotide analogs and is therefore exclusive to in-culturing living cells, and incorporation of nucleoside analogs into newly synthesized transcripts is prone to producing bias where RNA physically interacts with protein23,24. In iCLIP, only a single adapter is ligated to the 3&#39; extremity of crosslinked RNA fragments. After reverse transcription (RT), both truncated and read-through cDNAs are obtained by intramolecularly circularization and re-linearization followed by polymerase chain reaction (PCR) amplification25,26. However, the efficiency of intramolecular circularization is relatively low. Although older CLIP protocols need labeling of crosslinked RNA with a radioisotope, ultraviolet crosslinking and affinity purification (uvCLAP), with a process of stringent tandem affinity purification, does not rely on radioactivity27. Nevertheless, uvCLAP is limited to cultured cells that must be transfected with the expression vector carrying the 3x FLAG-HBH tag for tandem affinity purification.
In eCLIP, adapters were ligated first at the 3&#39; extremity of RNA followed by RT, and next at the 3&#39; extremity of cDNAs in an intermolecular mode. Hence, eCLIP is able to capture all truncated and read-through cDNA28. Also, it is neither restricted to radioactive labeling, nor to using cell lines based on its principle, while maintaining single-nucleotide resolution.
Here, we provide a step-by-step description of an eCLIP protocol adapted for mouse testis. Briefly, this eCLIP protocol starts with UV crosslinking of testicular tubules, followed by partial RNase digestion and immunoprecipitation using a protein-specific antibody. Next, the protein-bound RNA is dephosphorylated, and adapter is ligated to its 3&#39; end.&#160;After protein gel electrophoresis and gel-to-membrane transfer, RNA is isolated by cutting the membrane area of an expected size range. After RT, DNA adapter is ligated to the 3&#39; end of cDNA followed by PCR amplification. Screening of subclones prior to high-throughput sequencing is taken as a library quality control. This protocol is efficient at identifying major species of protein-bound RNA of RBPs, exemplified by the two testis-expressing RNA helicases MOV10L1 and MOV10.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse MOV10&amp;nbsp; antibody</td><td>Proteintech, China</td><td>10370-1-AP</td><td></td></tr><tr><td>Anti-mouse MOV10L1 antibody</td><td>Zheng et al.2010&lt;sup&gt;9&lt;/sup&gt;</td><td>polyclonal antisera UP2175</td><td>provided by P. Jeremy Wang lab, University of Pennsylvania</td></tr><tr><td>HRP Goat Anti-Rabbit IgG&amp;nbsp;</td><td>ABclonal</td><td>AS014</td><td></td></tr><tr><td>Rabbit IgG</td><td>Beyotime, China</td><td>A7016</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hamburg, Germany</td><td>5242R</td><td></td></tr><tr><td>Digital sonifier</td><td>BRANSON,USA</td><td>BBV12081048A</td><td>450 Watts; 50/60 Hz</td></tr><tr><td>DynaMag-2 Magnet</td><td>Invitrogen,USA</td><td>12321D</td><td></td></tr><tr><td>Mini Blot Module</td><td>Invitrogen,USA</td><td>B1000</td><td></td></tr><tr><td>Mini Gel Tank</td><td>Invitrogen,USA</td><td>A25977</td><td></td></tr><tr><td>Shaking incubator</td><td>Eppendorf, Hamburg, Germany</td><td>Thermomixer comfort&amp;nbsp;</td><td></td></tr><tr><td>Tissue Grinder, Dounce</td><td>PYREX, USA</td><td>1234F35</td><td>only&amp;nbsp; the &quot;loose&quot; pestle is used in this protocol</td></tr><tr><td>TProfessional standard 96 Gradient</td><td>Biometra, Germany</td><td>serial no.: 2604323</td><td></td></tr><tr><td>Tube Revolver&amp;nbsp;</td><td>Crystal, USA</td><td>serial no.: 3406051</td><td></td></tr><tr><td>UV-light cross-linker</td><td>UVP, USA</td><td>CL-1000</td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TC-treated Culture Dish</td><td>Corning, USA</td><td>430167</td><td>100 mm&amp;nbsp;</td></tr><tr><td>Tubes</td><td>Corning, USA</td><td>430791</td><td>15 mL</td></tr><tr><td>Microtubes tubes</td><td>AXYGEN , USA</td><td>MCT-150-C</td><td>1.5 mL&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acid phenol/chloroform/isoamyl alcohol&amp;nbsp;</td><td>Solarbio, China</td><td>P1011</td><td>25:24:01</td></tr><tr><td>AffinityScript Enzyme</td><td>Agilent, USA</td><td>600107</td><td></td></tr><tr><td>Antioxidant</td><td>Invitrogen,USA</td><td>NP0005</td><td></td></tr><tr><td>DH5&amp;alpha; competent bacteria</td><td>Thermo Scientific, USA</td><td>18265017</td><td>these economical cells yield &gt;1 x 10&lt;sup&gt;6&lt;/sup&gt; transformants/&amp;micro;g control DNA per 50 &amp;micro;L reaction.</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich, USA</td><td>D8418</td><td></td></tr><tr><td>DNA Ladder</td><td>Invitrogen, USA</td><td>10416014</td><td></td></tr><tr><td>dNTP</td><td>Sigma-Aldrich, USA</td><td>DNTP100-1KT</td><td></td></tr><tr><td>Dynabeads Protein A&amp;nbsp;</td><td>Invitrogen, USA</td><td>10002D</td><td></td></tr><tr><td>ECL reagent</td><td>Vazyme, China</td><td>E411-04</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen, USA</td><td>AM9260G</td><td></td></tr><tr><td>EDTA free protease inhibitor cocktail</td><td>Roche, USA</td><td>04693132001</td><td>add fresh</td></tr><tr><td>Exo-SAP-IT</td><td>Affymetrix, USA</td><td>78201</td><td>PCR Product Cleanup Reagent&amp;nbsp;</td></tr><tr><td>FastAP enzyme</td><td>Thermo Scientific, USA</td><td>EF0652</td><td></td></tr><tr><td>LDS Sample Buffer</td><td>Thermo Scientific, USA</td><td>NP0007</td><td></td></tr><tr><td>MetaPhor Agarose&amp;nbsp;</td><td>lonza, Switzerland</td><td>50180</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Invitrogen, USA</td><td>AM9530G</td><td></td></tr><tr><td>MiniElute gel Extraction</td><td>QIAGEN, Germany</td><td>28604</td><td>column store at 4 ?; buffer QG=gel dissolving buffer; buffer PE= wash buffer(for step 14)</td></tr><tr><td>MyONE Silane beads</td><td>Thermo Scientific, USA</td><td>37002D</td><td>nucleic acids extraction magnetic beads</td></tr><tr><td>NaCl</td><td>Invitrogen,USA</td><td>AM9759&lt;br/&gt; &amp;nbsp;</td><td></td></tr><tr><td>NP-40</td><td>Amresco, USA</td><td>M158-500ML</td><td></td></tr><tr><td>NuPAGE Bis-Tris Protein Gels</td><td>Invitrogen, USA</td><td>NP0336BOX</td><td>4%&amp;ndash;12%,1.5 mm, 15-well</td></tr><tr><td>NuPAGE MOPS SDS Buffer Kit&amp;nbsp;</td><td>Invitrogen, USA</td><td>NP0050</td><td></td></tr><tr><td>PBS</td><td>Gibco, USA</td><td>10010023</td><td></td></tr><tr><td>Phase-Locked Gel (PLG) heavy tube&amp;nbsp;&amp;nbsp;</td><td>TIANGEN, China</td><td>WM5-2302831</td><td></td></tr><tr><td>PowerUp SYBR Green Master Mix</td><td>Applied Biosystems, USA</td><td>A25742</td><td></td></tr><tr><td>Proteinase K</td><td>NEB, New England&amp;nbsp;</td><td>P8107S</td><td></td></tr><tr><td>Q5 PCR master mix&amp;nbsp;</td><td>NEB, New England&amp;nbsp;</td><td>M0492L</td><td></td></tr><tr><td>RLT buffer</td><td>QIAGEN, Germany</td><td>79216</td><td>RNA purification lysis buffer&amp;nbsp;</td></tr><tr><td>RNA Clean &amp;amp; Concentrator-5 columns&amp;nbsp;</td><td>ZYMO RESEARCH, USA</td><td>R1016</td><td>RNA purification and concentration columns</td></tr><tr><td>RNase I&amp;nbsp;</td><td>Invitrogen, USA</td><td>AM2295</td><td></td></tr><tr><td>RNase Inhibitor</td><td>Promega, USA</td><td>N251B</td><td></td></tr><tr><td>RQ1 DNase</td><td>Promega,USA</td><td>M610A</td><td></td></tr><tr><td>Sample Reducing Agent</td><td>Invitrogen,USA</td><td>NP0009</td><td></td></tr><tr><td>SDS Solution</td><td>Invitrogen, USA</td><td>15553027</td><td>10%</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich, USA</td><td>30970</td><td>protect from light</td></tr><tr><td>T4 PNK enzyme</td><td>NEB, New England&amp;nbsp;</td><td>M0201L</td><td></td></tr><tr><td>T4 RNA ligase 1 high conc</td><td>NEB, New England&amp;nbsp;</td><td>M0437M</td><td></td></tr><tr><td>TA/Blunt-Zero Cloning Mix</td><td>Vazyme, China</td><td>C601-01</td><td></td></tr><tr><td>TBE</td><td>Invitrogen,USA</td><td>AM9863</td><td></td></tr><tr><td>Tris-HCI Buffer</td><td>Invitrogen, USA</td><td>15567027</td><td></td></tr><tr><td>Triton X-100</td><td>Sangon Biotech, China</td><td>A600198&amp;nbsp;</td><td></td></tr><tr><td>Tween-20</td><td>Sangon Biotech, China</td><td>A600560</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich, USA</td><td>U5378</td><td></td></tr><tr><td>X-ray Films</td><td>Caresteam, Canada</td><td>6535876</td><td></td></tr></tbody></table>

enhanced crosslinking immunoprecipitation (eclip) method for efficient identification of protein-bound rna in mouse testis

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, underlining the importance of post-transcriptional regulation of gene expression orchestrated by RBPs. To elucidate mechanistic roles of an RBP, crosslinking immunoprecipitation (CLIP) methodology can be used to capture its endogenous direct RNA targets and define the actual interaction sites. The enhanced CLIP (eCLIP) is a newly-developed method that offers several advantages over the conventional CLIPs. However, the use of eCLIP has so far been limited to cell lines, calling for expanded applications. Here, we employed eCLIP to study MOV10 and MOV10L1, two known RNA-binding helicases, in mouse testis. As expected, we find that MOV10 predominantly binds to 3' untranslated regions (UTRs) of mRNA and MOV10L1 selectively binds to Piwi-interacting RNA (piRNA) precursor transcripts. Our eCLIP method allows fast determination of major RNA species bound by various RBPs via small-scale sequencing of subclones and thus availability of qualified libraries, as a warrant for proceeding with deep sequencing. This study establishes an applicable basis for eCLIP in mammalian testis.

The eCLIP procedure and results are illustrated in Figure 1, Figure 2, Figure 3, Figure 4. Mice were euthanized with carbon dioxide and a small incision was made in the lower abdomen using surgical scissors (Figure 2A,B). Mo...

Watch this Scientific Journal Video about Enhanced Crosslinking Immunoprecipitation eCLIP Method for Efficient Identification of Protein-bound RNA in Mouse Testis at JoVE.com

Enhanced Crosslinking Immunoprecipitation eCLIP Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Here, we present an eCLIP protocol to determine major RNA targets of RBP candidates in testis.

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, ...

enhanced-crosslinking-immunoprecipitation-eclip-method-for-efficient

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

19.8K Views.  Nanjing Medical University. Here, we present an eCLIP protocol to determine major RNA targets of RBP candidates in testis.

Video: Enhanced Crosslinking Immunoprecipitation eCLIP Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Genetics

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Biochemistry

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)&#8212;we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-&#954;B pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers&#8212;as it portends a significant roadblock against immunotherapy&#8212;the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, ...

Developmental Biology

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions

Source: Singh, R. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59635">Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins.</a> J. Vis. Exp. ( (2019).
In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis

Summary

Explore More Videos

Chapters in this video

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TE^deff) in Cancers

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

SELEX-Based In Vitro Binding Assay to Identify RNA-Protein Interactions