This work was made possible by the&#160;NIH R01 grant (NS094721, K.A.J.).&#160;

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The authors declare no conflict of interest.

In in vitro SHAPE, it is critical to use high quality homogeneous RNA template. T7 transcription, however, often yields heterogeneous sequences36. Especially, sequences with &#177;1 nucleotide at the 3&#39;-terminus with non-negligible yields36 are usually difficult to be removed by polyacrylamide gel purification. Heterogeneous RNA template can result in more than one set of the signal in the sequencing gel profiling of the primer extension product, which sometimes makes i...

Selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) is a method of measuring the kinetics of each nucleotide in an RNA sequence of interest and elucidating the secondary structure at single-nucleotide resolution1. SHAPE methodologies, both in in vitro conditions2,3,4 (purified RNA in a defined buffer system) and in living mammalian cells5,6, have been developed to investigate the secondary structure of medium length RNA sequences (typically &#60;1,000 nucleotides for in-cell SHAPE and &#60;200 nucleotides for in vitro SHAPE). It is particularly useful to evaluate structural changes in receptor RNA upon binding to RNA-interacting small molecule metabolites2,4,7,8 and to study mechanistic actions of RNA-targeting molecules during drug development9,10.
RNA-targeting drug discovery has recently drawn attention in academic laboratories and the pharmaceutical industry11,12 via different approaches and strategies13,14,15,16. Recent examples of RNA-targeting small molecules for clinical use include two structurally distinct experimental drugs, LMI-07017 and RG-791618,19, for spinal muscular atrophy (SMA), which showed promising results in phase II clinical trials20. Both molecules were demonstrated to target survival of motor neuron (SMN) 2 pre-mRNA and regulate the splicing process of the SMN2 gene6,17,21. We previously demonstrated the application of in vitro and in-cell SHAPE in an examination of the target RNA structural changes in the presence of an analog of RG-7916 known as SMN-C26.
In principle, SHAPE measures the 2&#39;-OH acylation rate of each nucleotide of an RNA sequence in the presence of excess amounts of a self-quenching acylation reagent in an unbiased manner. The acylation reagent is not stable in water, with a short half-life of (e.g., T1/2 = 17 s for 1-methyl-7-nitroisatoic anhydride; or 1M7, ~20 min for 2-methylnicotinic acid imidazolide, or NAI)22 and insensitivity to the identity of the bases23. This results in a more favorable acylation of the 2&#39;-OH groups of flexible bases, which can be transformed into an accurate assessment of the dynamics of each nucleotide. Specifically, a nucleotide in a base-pair is usually less reactive than an unpaired one to a 2&#39;-OH modifying reagent, such as NAI and 1M7.
Looking at the source of the RNA template and where 2&#39;-OH acylation takes place, SHAPE can generally be categorized into in vitro and in-cell SHAPE. In vitro SHAPE uses purified T7 transcribed RNA and lacks a cellular context in experimental designs. In in-cell SHAPE, both the RNA template transcription and 2&#39;-OH acylation occur within living cells; therefore, the results can recapitulate the RNA structural model in a cellular context. In-cell SHAPE has been referred to as in vivo SHAPE for the SHAPE carried in living cells in the literature24. Since this experiment is not performed in an animal, we termed this experiment as in-cell SHAPE for accuracy.
The strategies for the primer extension stage of in vitro and in-cell SHAPE are also different. In in vitro SHAPE, reverse transcription stops at the 2&#39;-OH acylation position in the presence of Mg2+. A 32P-labled primer extension therefore appears as a band in polyacrylamide gel electrophoresis (PAGE) and the intensity of the band is proportional to the acylation rate1. In in-cell SHAPE, reverse transcription generates random mutations at the 2&#39;-OH adduct position in the presence of Mn2+. The mutational rate of each nucleotide can be captured by in depth next-generation sequencing, and the SHAPE reactivity at single-nucleotide resolution can then be calculated.
A potential problem for in-cell SHAPE is the low signal-to-noise ratio (i.e., a majority of the 2&#39;-OH groups is unmodified, while the unmodified sequences occupy most of the read in next-generation sequencing). Recently, a method to enrich the 2&#39;-OH modified RNA, referred to as in vivo click SHAPE (icSHAPE), was developed by the Chang laboratory25. This enrichment method may be advantageous in studying weak small molecules such as RNA interactions, especially in a transcriptome-wide interrogation.

<table><tbody><tr><td>DNA oligonucleotide</td><td>IDT</td><td></td><td>gBlock for &gt;200 bp DNA synthesis</td></tr><tr><td>Phusion Green Hot Start II High-Fidelity PCR Master Mix</td><td>Thermo Fisher</td><td>F566S</td><td></td></tr><tr><td>NucleoSpin gel and PCR clean-up kit</td><td>Takara</td><td>740609.50</td><td></td></tr><tr><td>MegaScript T7 transcription kit</td><td>Thermo Fisher</td><td>AM1333</td><td>Contains 10x reaction buffer, T7 enzyme, NTP and Turbo DNase</td></tr><tr><td>DEPC-treated water</td><td>Thermo Fisher</td><td>750023</td><td></td></tr><tr><td>2x TBE-urea sample buffer</td><td>Thermo Fisher</td><td>LC6876</td><td></td></tr><tr><td>40% acrylamide/ bisacrylamide solution (29:1)</td><td>Bio-Rad</td><td>1610146</td><td></td></tr><tr><td>10x TBE buffer</td><td>Thermo Fisher</td><td>15581044</td><td></td></tr><tr><td>Nalgene Rapid-Flow&amp;trade; Filter Unit</td><td>Thermo Fisher</td><td>166-0045</td><td></td></tr><tr><td>Kimwipe</td><td>Kimberly-Clark</td><td>34133</td><td></td></tr><tr><td>TEMED</td><td>Thermo Fisher</td><td>17919</td><td></td></tr><tr><td>SYBR-Safe dye</td><td>Thermo Fisher</td><td>S33102</td><td></td></tr><tr><td>6% TBE-urea mini-gel</td><td>Thermo Fisher</td><td>EC6865BOX</td><td></td></tr><tr><td>ChemiDoc</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>T4 PNK</td><td>NEB</td><td>M0201S</td><td></td></tr><tr><td>&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P-ATP</td><td>Perkin Elmer</td><td>NEG035C005MC</td><td></td></tr><tr><td>Hyperscreen&amp;trade; Intensifying Screen</td><td>GE Healthcare</td><td>RPN1669</td><td>calcium tungstate phosphor screen</td></tr><tr><td>phosphor storage screen</td><td>Molecular Dynamics</td><td>BAS-IP MS 3543 E</td><td></td></tr><tr><td>Amersham Typhoon</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>NAI (2M)</td><td>EMD Millipore</td><td>03-310</td><td></td></tr><tr><td>GlycoBlue</td><td>Thermo Fisher</td><td>AM9515</td><td></td></tr><tr><td>SuperScript IV Reverse Transcriptase</td><td>Thermo Fisher</td><td>18090010</td><td>Contains 5x RT buffer, SuperScript IV</td></tr><tr><td>dNTP mix (10 mM)</td><td>Thermo Fisher</td><td>R0192</td><td></td></tr><tr><td>ddNTP set (5 mM)</td><td>Sigma</td><td>GE27-2045-01</td><td></td></tr><tr><td>large filter paper</td><td>Whatman</td><td>1001-917</td><td></td></tr><tr><td>Gel dryer</td><td>Hoefer</td><td>GD 2000</td><td></td></tr><tr><td>QIAamp DNA Blood Mini Kit</td><td>Qiagen</td><td>51104</td><td>Also contains RNase A and protease K</td></tr><tr><td>SMN2 minigene&lt;sup&gt;34&lt;/sup&gt;</td><td>Addgene</td><td>72287</td><td></td></tr><tr><td>Heat inactivated FBS</td><td>Thermo Fisher</td><td>10438026</td><td></td></tr><tr><td>Pen-Strep</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>Opti-MEM I</td><td>Thermo Fisher</td><td>31985062</td><td></td></tr><tr><td>FuGene HD</td><td>Promega</td><td>E2311</td><td></td></tr><tr><td>TrpLE</td><td>Thermo Fisher</td><td>12605010</td><td></td></tr><tr><td>DPBS without Ca/Mg</td><td>Thermo Fisher</td><td>14190250</td><td></td></tr><tr><td>TRIzol</td><td>Thermo Fisher</td><td>15596018</td><td></td></tr><tr><td>RNeasy mini column</td><td>Qiagen</td><td>74104</td><td>Also contains RW1, RPE buffer</td></tr><tr><td>RNase-Free DNase Set</td><td>Qiagen</td><td>79254</td><td>Contains DNase I and RDD buffer</td></tr><tr><td>Deionized formamide</td><td>Thermo Fisher</td><td>AM9342</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M3634</td><td></td></tr><tr><td>random nonamer</td><td>Sigma-Aldrich</td><td>R7647</td><td></td></tr><tr><td>SuperScript First-Strand Synthesis System</td><td>Thermo Fisher</td><td>11904-018</td><td>Contains 10x RT buffer, SuperScript II reverse transcriptase</td></tr><tr><td>AccuPrime pfx DNA polymerse</td><td>Thermo Fisher</td><td>12344024</td><td></td></tr><tr><td>NextSeq500</td><td>Illumina</td><td></td><td></td></tr><tr><td>NucAway column</td><td>Thermo Fisher</td><td>AM10070</td><td>for desalting purpose</td></tr></tbody></table>

using in vitro and in-cell shape to investigate small molecule induced pre-mrna structural changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

We previously demonstrated that an RNA splicing modulator, SMN-C2, interacts with AGGAAG motif on exon 7 of the SMN2 gene&#39;s pre-mRNA, and used SHAPE to assess the RNA structural changes in the presence of SMN-C26. The binding site of SMN-C2 is distinct from the FDA-approved antisense oligonucleotide (ASO) for SMA, nusinersen, which binds and blocks the intronic splicing silencer (ISS) on intron 727,28 (...

Watch this Scientific Journal Video about Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes at JoVE.com

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

using-vitro-cell-shape-to-investigate-small-molecule-induced-pre-mrna

Universidad San Sebastian

Research

JoVE Journal

Genetics

8.2K Views.  The Scripps Research Institute. Detailed protocols for both in vitro and in-cell selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

Video: Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Biochemistry

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

Cancer Research

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Biology

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

Summary

Explore More Videos

Chapters in this video

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

Using the E1A Minigene Tool to Study mRNA Splicing Changes

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes