Dr Ren&#39;s work is supported by the American Heart Association (13SDG14800019; BR), the Ann&#39;s Hope Foundation (FP00011709; BR), the American Cancer Society (86-004-26; the MCW Cancer Center to BR), and the National Institute of Health (HL136423; BR); Jordan Palmer is supported by the 2018 MCW CTSI 500 Stars Internship Program; P. Moran is supported by an Institutional Research Training Grant from NHLBI (5T35 HL072483-34).

For animal experiments, all methods described here have been approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.
1. Prepare reagents
<ol>
	<li>Prepare lysis buffer to concentrations of 10 mM HEPES, pH 7.4, 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 100 &#181;g/mL cycloheximide, protease inhibitors, and recombinant RNase inhibitors to concentrations as described below.
	<ol>
		<li>Add following reagents to 500 mL of RNase-free deionized water: 1.19 g of HEPES, 5.59 g of KCl, 0.24 g of MgCl2, 35 mg of DTT, 0.5 mL of cycloheximide, and NaOH as needed until pH 7.4, EDTA-free protease inhibitors (one mini tablet per 10 mL) and RNase inhibitor (10 &#181;L/mL).</li>
		<li>Store in a 4 &#176;C fridge for up to 1 month.</li>
	</ol>
	</li>
	<li>Prepare a high-salt polysome wash buffer to concentrations of 10 mM HEPES, pH 7.4, 350 mM KCl, 5 mM MgCl2, 1% vol/vol CA-630, 0.5 mM DTT, and 100 &#181;g/mL cycloheximide.
	<ol>
		<li>Add following reagents to 500 mL of RNase-free deionized water: 1.19 g of HEPES, 13.05 g of KCl, 0.24 g of MgCl2, 5 mL of nonionic, non-denaturing detergent, 5 of 7.7 mg tubes of DTT, and 0.5 mL of cycloheximide, and NaOH as needed until pH 7.4.</li>
		<li>Store in 4 &#176;C fridge for up to 1 month.</li>
	</ol>
	</li>
	<li>Bind anti-GFP antibody to Protein G magnetic beads prior to starting experiment.
	<ol>
		<li>Add 10 &#181;g of anti-GFP antibody diluted in 200 &#181;L of PBS to Protein G beads.</li>
		<li>Incubate with end over end rotation for 10 minutes at room temperature.</li>
		<li>Place the beads on a magnetic rack and remove the supernatant.</li>
		<li>Suspend the beads in 200 &#181;L of PBS and store in 4 &#176;C fridge for up to 1 week.</li>
	</ol>
	</li>
	<li>Prepare ice-cold PBS with 100 &#181;g/mL cycloheximide.
	<ol>
		<li>Add 1 volume of cycloheximide solution (100 mg/mL) to 999 volumes of ice-cold PBS.</li>
	</ol>
	</li>
</ol>
2. Isolate and lyse desired tissues
<ol>
	<li>Euthanize mice by IP injection of ketamine (500 mg/kg/body weight) and xylazine (10 mg/kg/body weight) and isolate desired tissues (i.e., heart, lung). Immediately proceed to next step.</li>
	<li>Place desired tissues into 500 &#181;L of ice-cold PBS with 100 &#181;g/mL cycloheximide.</li>
	<li>Mince tissue into a cell suspension with a motor-driven homogenizer or a small-clearance glass homogenizer. If using a motor-driven homogenizer, limit homogenization to less than 1 minute at low frequency (&#60;15,000 Hz) to avoid RNA denaturation.</li>
	<li>Suspend cell pellet in 200 &#181;L of lysis buffer by pipetting and redrawing up buffer several times. Further homogenize cell suspension with 10 strokes in a small-clearance glass homogenizer or for 15 seconds at low frequency (&#60;15,000 Hz) in a motor driven homogenizer.</li>
	<li>Centrifuge homogenates for 10 min at 2,000 x g at 4 &#176;C to pellet nuclei and large cell debris, and keep the supernatant.</li>
	<li>Add nonionic, non-denaturing detergent to 1% vol/vol and DHPC to 30 mM to the supernatant. Incubate on ice for 5 min.</li>
	<li>Centrifuge lysate for 10 min at 16,000 x g to pellet insoluble material. Transfer and keep 15% of clear lysate as input for future steps.</li>
</ol>
3. Isolate ribosome/mRNA complexes
<ol>
	<li>Add 50 &#181;L of antibody-bound beads to cell-lysate supernatant and incubate mixture at 4 &#176;C with end-over-end rotation for 30 min. This is where the anti-GFP antibodies will bind the GFP-tagged ribosomes, allowing us to further isolate the RNA from these ribosomes.</li>
	<li>Collect beads on a magnetic rack and wash 5 times with high-salt polysome wash buffer.
	<ol>
		<li>Draw up and discard liquid once beads have collected on the side of the tube. Then pipette and redraw up 200 &#181;L of high-salt polysome wash buffer several times. Repeat this step 5 times and discard all buffer following final repetition. Immediately proceed to next step.</li>
	</ol>
	</li>
</ol>
4. Isolate mRNA
<ol>
	<li>Place beads in RLT buffer. The following steps are taken directly from the RNeasy mini kit protocol and were not expanded on in any way. 
	CAUTION: RLT buffer contains guanidine salts; do NOT mix with bleach.</li>
	<li>Centrifuge lysate for 3 min at full speed 13,000 rpm or 16,000 g at 4 &#176;C. Carefully remove supernatant of 350 &#181;L by pipetting and transfer it to a new microfuge tube. Use only this supernatant (lysate) in subsequent steps.</li>
	<li>Add an equal volume of 70% ethanol into the microfuge tube.</li>
	<li>Transfer up to 700 &#181;L of the sample, including any precipitate that may have formed, to a spin column placed in a 2 mL collection tube. Close the lid gently and centrifuge for 15 s at &#8805;8,000 x g to wash the spin column membrane. Discard the flow-through.</li>
	<li>Add 350 &#181;L of buffer RW1 to the spin column. Close the lid gently and centrifuge for 15 s at &#8805;8,000 x g to wash the spin column membrane. Discard the flow-through and reuse the collection tube in next step. 
	CAUTION: Buffer RW1 contains guanidine salts; do NOT mix with bleach.</li>
	<li>Add 350 &#181;L of buffer RW1 to the spin column. Close the lid gently and centrifuge for 15 s at &#8805;8,000 x g. Discard the flow-through.</li>
	<li>Add 500 &#181;L of buffer RPE to the spin column. Close the lid gently and centrifuge for 15 s at &#8805;8,000 x g to wash the spin column membrane. Discard the flow-through.</li>
	<li>Add 500 &#181;L of buffer RPE to the spin column. Close the lid gently and centrifuge for 2 min at &#8805;8,000 x g to wash the spin column membrane. Then carefully remove the spin column from the collection tube, ensuring that the column does not contact the flow-through.</li>
	<li>Place the spin column in a new 2 mL collection tube and discard the old tube with the flow-through. Close the lid gently and centrifuge at full speed for 1 min to remove residual buffer.</li>
	<li>Place the spin column in a new 1.5 mL collection tube. Add 30-50 &#181;L of RNase-free water directly to the spin column membrane. Close the lid gently and centrifuge for 1 min at &#8805;8,000 x g to elute the RNA.</li>
	<li>If expected RNA yield is &#62;30 &#181;g, repeat step 4.10 with another 30-50 &#181;L of RNase-free water, or using elute from Step 4.10 (if high [RNA] is required). Reuse collection tube from Step 4.10.</li>
	<li>Use purified RNA for downstream analysis including RNA-sequencing or real time quantitative PCR or store RNA dissolved in RNase-free H2O at -80 &#176;C for up to 1 year.</li>
</ol>

<ol>
	<li>Heiman, M., Kulicke, R., Fenster, R. J., Greengard, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+mRNA+purification+by+translating+ribosome+affinity+purification+(TRAP).">Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).</a> Nature Protocols. 9, 1282-1291 (2014).</li><li>Zhou, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrogating+translational+efficiency+and+lineage-specific+transcriptomes+using+ribosome+affinity+purification.">Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 15395-15400 (2013).</li><li>Best, B., Moran, P., Ren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF/PKD-1+signaling+mediates+arteriogenic+gene+expression+and+angiogenic+responses+in+reversible+human+microvascular+endothelial+cells+with+extended+lifespan.">VEGF/PKD-1 signaling mediates arteriogenic gene expression and angiogenic responses in reversible human microvascular endothelial cells with extended lifespan.</a> Molecular and Cellular Biochemistry. 446, 199-207 (2018).</li><li>Ren, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPA/PKD-1-FoxO1+Signaling+Axis+Mediates+Endothelial+Cell+CD36+Transcriptional+Repression+and+Proangiogenic+and+Proarteriogenic+Reprogramming.">LPA/PKD-1-FoxO1 Signaling Axis Mediates Endothelial Cell CD36 Transcriptional Repression and Proangiogenic and Proarteriogenic Reprogramming.</a> Arteriosclerosclerosis Thrombosis, Vascular Biology. 36, 1197-1208 (2016).</li><li>Ren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Kinase+D1+Signaling+in+Angiogenic+Gene+Expression+and+VEGF-Mediated+Angiogenesis.">Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis.</a> Frontiers in Cell and Developmental Biology. 4, 37 (2016).</li><li>Ren, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxO1+transcriptional+activities+in+VEGF+expression+and+beyond:+a+key+regulator+in+functional+angiogenesis?.">FoxO1 transcriptional activities in VEGF expression and beyond: a key regulator in functional angiogenesis?.</a> Journal of Pathology. 245, 255-257 (2018).</li><li>Hupe, M., Li, M. X., Gertow Gillner, K., Adams, R. H., Stenman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+TRAP-sequencing+technology+with+a+versatile+conditional+mouse+model.">Evaluation of TRAP-sequencing technology with a versatile conditional mouse model.</a> Nucleic Acids Research. 42, e14 (2014).</li><li>Dong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+obesity+links+to+ER+positive+breast+cancer+progression+via+LPA/PKD-1-CD36+signaling-mediated+microvascular+remodeling.">Diet-induced obesity links to ER positive breast cancer progression via LPA/PKD-1-CD36 signaling-mediated microvascular remodeling.</a> Oncotarget. 8, 22550-22562 (2017).</li><li>Ren, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK1/2-Akt1+crosstalk+regulates+arteriogenesis+in+mice+and+zebrafish.">ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish.</a> Journal of Clinical Investigation. 120, 1217-1228 (2010).</li><li>Skuli, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+HIF-2alpha+regulates+murine+pathological+angiogenesis+and+revascularization+processes.">Endothelial HIF-2alpha regulates murine pathological angiogenesis and revascularization processes.</a> Journal of Clinical Investigation. 122, 1427-1443 (2012).</li></ol>

The authors declare that they have no conflict of interest.

Angiogenesis is a complex multistep process, in which EC-specific angiogenic gene transcription and expression play an essential role in EC differentiation and angiogenic reprogramming3,4. To overcome the barriers from the cellular diversity and architectural complexity for better understanding the function of the mammalian vascular system at a molecular level in vivo, we have created EC-specific TRAP mice, accompanied by EC-specific cd36, EC-specific <e...

In complex tissues such as the mammalian brain, heart and lung, the high levels of cellular heterogeneity complicate the analysis of gene expression data derived from whole tissue samples. To observe gene expression profiles in a particular cell type in vivo, a new methodology has been developed recently, which allows the interrogation of the entire translated mRNA complement of any genetically defined cell type. This methodology is known as the translating ribosome affinity purification (TRAP) technique1,2. It is a useful tool to study endothelial cell biology and angiogenesis when combined with genetically manipulating other angiogenesis-associated genes in animals.
We have shown that angiogenic PKD-1 signaling and the transcription of angiogenic gene CD36 are critical for endothelial cell (EC) differentiation and functional angiogenesis3,4,5,6. To determine molecular mechanisms of angiogenic and metabolic signaling in gene transcription and EC transdifferentiation, we have created genetically engineered TRAP mice with specifically deleted angiogenic genes on the basis of TRAP technique1,2. Furthermore, in our TRAP animals, not only do they have pkd-1 or cd36 gene deficiency in the vascular endothelia or global deletion of cd36 gene, but an enhanced green fluorescence protein (EGFP) is also genetically tagged onto EC&#39;s translating ribosomes. TRAP permits affinity purification of ribosome-bound mRNA directly from the vascular endothelia of targeted tissues, enabling the analysis of gene expression and identification of new transcriptomes that are associated with EC differentiation and angiogenesis directly under in vivo conditions. We have successfully isolated ribosome-bound RNA from the endothelia in these genetically engineered animals. The purified RNA can be used for further characterization of angiogenic or arteriogenic genes in the regulation of EC differentiation and functions. This protocol provides a step-by-step guide to implement the TRAP approach for the isolation of mRNA in ECs directly in vivo.

<table><tbody><tr><td>2100 Electrophoresis Bioanalyzer with Nanochips and Picochips</td><td>Agilent</td><td>G2939AA, 5067-1511 &amp;amp; 5067-1513</td><td></td></tr><tr><td>Cell scrapers</td><td>Sarstedt</td><td>83.1832</td><td></td></tr><tr><td>Homogenizers</td><td>Fisher Scientific</td><td>K8855100020</td><td></td></tr><tr><td>Magnet (Dynamag-2)</td><td>Invitrogen</td><td>123-21D</td><td>Will depend on purification scale; samples in 1.5-mL tubes can be concentrated on a DynaMag-2</td></tr><tr><td>Minicentrifuge</td><td>Fisher Scientific</td><td>05-090-100</td><td></td></tr><tr><td>NanoDrop 2000C spectrophotometer</td><td>Thermo Scientific&amp;nbsp;</td><td>ND-2000C</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5430R</td><td>with rotor for 1.5-mL microcentrifuge tubes</td></tr><tr><td>RNase-free 1.5mL microcentrifuge tubes</td><td>Applied Biosystems&amp;nbsp;</td><td>AM12450</td><td></td></tr><tr><td>Rnase-free 50-mL conical tubes</td><td>Applied Biosystems&amp;nbsp;</td><td>AM12501</td><td></td></tr><tr><td>RNase-free 1000-&amp;mu;l filter tips</td><td>Rainin</td><td>RT-1000F</td><td></td></tr><tr><td>RNase-free 200-&amp;mu;l filter tips</td><td>Rainin&amp;nbsp;</td><td>RT-200F</td><td></td></tr><tr><td>RNase-free 20-&amp;mu;l filter tips</td><td>Rainin&amp;nbsp;</td><td>RT-20F</td><td></td></tr><tr><td>Rotor for homogenizers</td><td>Yamato&amp;nbsp;</td><td>LT-400D</td><td></td></tr><tr><td>Tube rotator, Labquake brand</td><td>Thermo Fisher</td><td>13-687-12Q</td><td></td></tr></tbody></table>

translating ribosome affinity purification (trap) for rna isolation from endothelial cells in vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Our previous studies4,7 suggest that CD36 may function as a switch for arteriolar differentiation and capillary arterialization via the LPA/PKD-1 signaling pathway. To study whether the LPA/PKD-1-CD36 signaling axis is essential for arteriogenesis in vivo, we have established the novel TRAP lines that not only have global cd36 deficiency or endothelial-specific-cd36- or pkd-1-deficiency but also permit selective isolation of ribosome-bound RNA from cre-marked cel...

Watch this Scientific Journal Video about Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo at JoVE.com

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

translating-ribosome-affinity-purification-trap-for-rna-isolation

Research

JoVE Journal

Biochemistry

11.7K Views.  Blood Center of Wisconsin. We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Video: Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

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

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

Cancer Research

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

Neuroscience

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

Biology

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 ...

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

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Chapters in this video

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

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

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling