This work was supported by Wellcome funding to JB [104648]. Work in the Wellcome Centre for Cell Biology is supported by Wellcome core funding [092076]. The authors acknowledge members of the lab for their help: Bella Maudlin, Emanuela Sani, Susanna De Lucas-Arias and Shiney George. The authors would also like to thank Patrick Cramer for the plasmid YEpEBI31111.

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D., 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=RiboSys,+a+high-resolution,+quantitative+approach+to+measure+the+in+vivo+kinetics+of+pre-mRNA+splicing+and+3&#8242;-end+processing+in+Saccharomyces+cerevisiae.">RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3&#8242;-end processing in Saccharomyces cerevisiae.</a> RNA. 16, 2570-2580 (2010).</li><li>Wallace, E. W. J., Beggs, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extremely+fast+and+incredibly+close:+cotranscriptional+splicing+in+budding+yeast.">Extremely fast and incredibly close: cotranscriptional splicing in budding yeast.</a> RNA. 23, 601-610 (2017).</li><li>Adelman, K., Lis, J. 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The authors have nothing to disclose.

This article presents a protocol for extremely rapid and specific 4tU labelling, for recovery of nascent, newly synthesized RNA from S. cerevisiae after as little as 15 s of labelling, with very low contamination by unlabeled RNA. 
The user should always take care to maintain the integrity of the RNA by use of cold temperatures and DEPC-treated reagents. Streptavidin bead purification is generally reliable; however, the bead buffer is difficult to handle; it must be made freshly, with its components added in the right order, and not chilled or autoclaved. Common failings include the RNA being incompletely dissolved after the precipitation steps, and so being either not biotinylated or otherwise lost during the processing steps. There is extensive troubleshooting help in the supplementary material.
There are some limitations to be aware of in ers4tU. One already mentioned is that 4tU slows growth of the yeast (Figure 1a). Apart from endogenously thiolated RNAs9, only RNAs that have been transcribed during the labelling period can be purified by this method. Polymerases paused on genes throughout the thiolation time will not produce thiolated transcripts that can be purified, although transcripts that are partially labelled due to polymerases entering or leaving a paused state during thiolation can be recovered. Strains that transcribe poorly, either because of mutation or growth conditions, produce little nsRNA, although the techniques used here will nevertheless improve recovery of nsRNA compared to other methods. Longer times and increased culture volumes may be necessary in these strains and conditions. Note that uracil is a good source of nitrogen and so this method should be trialed before being used for studies involving nitrogen starvation.
The ers4tU protocol is particularly useful for analysis of short-lived RNAs, many of which are so rapidly degraded that they cannot be identified without crippling the degradation machinery. Examples include cryptic unstable transcripts (CUTs)4, and short transcripts produced by premature termination or promoter proximal pausing18 and antisense transcription "upstream" from a promoter (PROMPTs)19. The intermediates produced during processing of stable RNA species are also transient but can be enriched using ers4tU transcription4. The ers4tU protocol is therefore exceptional in permitting highly transient RNA species to be analyzed and captured under near physiological conditions, which is a huge advantage over other methods. This technique has been used to study transcription and downstream RNA processing kinetics in RNA polymerase mutants that elongate faster or slower than normal20.
Thiolation is also compatible with RNA-seq and SLAM-seq21, allowing all RNA produced within a very short time window to be characterized in exquisite detail.

RNA has a dynamic nature; soon after it is produced much RNA is rapidly processed and degraded. Currently, most studies of RNA metabolism analyze the total cellular RNA, which is mostly fully processed and at steady state level. This level depends on the balance between the rates of transcription, post-transcriptional maturation and degradation. Analysis of the processes that lead to the steady state equilibrium requires specialized techniques to capture very short-lived RNA species.
Metabolic labelling of RNA with nucleotide analogues such as 4-thiouracil (4tU) or 4-thiouridine (4sU) (see Duffy et al.1 for an excellent review), offers the ability to isolate thio-labelled nascent RNAs and their processing intermediates. However, published protocols involve labelling times of several minutes2,3, which is slow relative to the rate of production of many transcripts. It takes in the order of one minute to transcribe the average yeast gene, so labelling yeast RNA for less than one minute can be considered extremely short. The extremely rapid and specific 4 thiouracil protocol (ers4tU) maximizes the signal to noise ratio by maximizing 4tU incorporation and minimizing the recovery of unlabeled, pre-existing RNA making very short labelling times possible4.
The thio-modified base must be imported into the cells rapidly and in sufficient quantity to efficiently label the newly synthesized RNA (nsRNA). To promote this, cells are grown in uracil-free medium, and expression of an appropriate permease helps to boost 4tU or 4sU uptake (see Table 1 for a list of plasmids that carry suitable permease genes and Supplementary Figure 1). 4tU&#39;s solubility in sodium hydroxide avoids the need for toxic organic solvents required by other nucleotide analogues. Unfortunately, growing cultures for long periods with thio-modified nucleosides at concentrations greater than 50 &#181;M has been observed to disrupt ribosomes5. However, the concentration (10 &#181;M) used here, and the extremely short labelling times, minimize deleterious effects5 (Figure 1a), while still yielding sufficient RNA for analysis.
This technique can be combined with rapid and specific auxin-mediated depletion of a target protein6,7 (Figure 2), referred to as the &#34;&#946;-est AID 4U&#34; protocol, in which &#946;-estradiol regulated expression of the auxin inducible degron (AID) system is combined with 4tU labelling. With the &#946;-est AID 4U approach, a target protein can be depleted and the effect on RNA metabolism closely monitored (Figure 2). The timing is critical; it is advisable to view the accompanying video and pay close attention to Figure 2 and its animated form (see Supplementary Figure 2).
Processing and degradation of RNA must be stopped extremely rapidly for accurate time resolution. This is achieved using methanol at low temperature, which fixes the cell contents very rapidly and degrades the cell membrane while preserving the nucleic acid content8. The RNA extraction should be efficient and not damage the RNA. Mechanical lysis is effective in the absence of chaotropic agents (often these contain thio groups, so should be avoided). Lithium chloride precipitation of RNA is preferred, as tRNAs are less efficiently precipitated. tRNAs are rapidly transcribed and naturally thiolated9, so removing tRNAs reduces competition for the biotinylation reagent. If small, highly structured RNAs are of interest, alcohol-based RNA precipitation methods are recommended.
To recover the thiolated RNA, biotin is covalently attached via the thio groups incorporated into the RNA with 4tU. The use of modified biotin, which attaches via a cleavable disulfide bond (e.g., HPDP-biotin (N-[6-(Biotinamido)hexyl]-3&#180;-(2&#180;-pyridyldithio)propionamide, ) or MTS-biotin (Methane thiosulfonate)) is recommended as it permits release of the RNA by addition of a reducing agent. The biotinylated RNA is affinity purified on streptavidin coupled to magnetic beads. This protocol is similar to others listed previously10 but has been intensively optimized to reduce background.
There are two types of thiol-labelling experiment that can be performed, continuous and discontinuous labelling. Each has its own advantages. In continuous labelling the 4tU is added to the culture and samples taken at regular intervals. This type of experiment shows how the RNA is processed and how levels change over time. Examples include comparison of mutant with wild-type experiments and a pulse-chase experiment. The experiments shown in Figure 3b,c are of this type. For discontinuous labelling a change is induced into the system and the RNA monitored. Once the change has been induced the culture must be split into several sub-cultures, and at specific times, each one is then thio-labelled for a brief period. One example is &#946;-est AID 4U shown in Figure 27. This type of experiment is particularly useful for monitoring the effect of a metabolic change on RNA processing (see Figure 3d).
A graphical representation of a thio-labelling experiment is presented in Figure 4 and Figure 5, and a spreadsheet that greatly simplifies the performance of the protocol is available (see 4tU experiment template.xlsx). As well as this the Supplementary Information contains an extensive troubleshooting guide. For the &#946;-est AID 4U protocol that integrates 4tU labelling with the auxin depletion protocol, see Figure 2 and Supplementary Figure 2. See Barrass et al.7 for the detailed AID depletion protocol.

<table><tbody><tr><td>&amp;beta;-mercaptoethanol (&amp;beta;ME)</td><td>Sigma-Aldrich</td><td>M3148</td><td>CAUTION toxic. Stock solutions are aproximatly 14 M, make at &lt;sup&gt;1&lt;/sup&gt;/&lt;sub&gt;20&lt;/sub&gt; dilution for use</td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>25668</td><td>CAUTION toxic</td></tr><tr><td>Diethyl pyrocarbonate (DEPC)</td><td>Sigma-Aldrich</td><td>D5758</td><td>add &lt;sup&gt;1&lt;/sup&gt;/&lt;sub&gt;1000&lt;/sub&gt; volume to a solution, leave at room temperature for 24 h, then autoclave</td></tr><tr><td>DMF (N,N-dimethylformamide)</td><td>&amp;nbsp;Sigma-Aldrich</td><td>227056</td><td>CAUTION toxic</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>3609</td><td>Make 0.5 M and pH to 8.0 with sodium hydroxide</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>29221</td><td></td></tr><tr><td>EZ-link HPDP Biotin</td><td>Thermo scientific</td><td>21341</td><td>Store protected from light. Disolve all the vial contents in 22.7 mL DMF (to make a 4 mM stock solution). Store away from water, in the dark &amp;amp; at -20 &amp;deg;C. Check the solution before using, as some batches of HPDP precipitate in storage; heat at 42 &amp;deg;C to resuspend.</td></tr><tr><td>Glucose</td><td>Fisher Scientific&amp;nbsp;</td><td>G/0500/60</td><td></td></tr><tr><td>Glycogen [20 mg/mL]</td><td>Sigma-Aldrich</td><td>10901393001</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Immobilised TCEP Disulfide Reducing Gel</td><td>Thermo Scientific</td><td>77712</td><td>Optional</td></tr><tr><td>LiCl</td><td>Sigma-Aldrich</td><td>793620</td><td>10 M solution. CAUTION: this gets very hot as is dissolves and can even boil at greater than 100 &lt;sup&gt;o&lt;/sup&gt;C, add the LiCl crystals to the water slowly.</td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>63033</td><td>1 M solution. CAUTION: this gets very hot as is dissolves and can even boil at greater than 100 &lt;sup&gt;o&lt;/sup&gt;C, add the MgCl&lt;sub&gt;2&lt;/sub&gt; crystals to the water slowly.</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>M/4000/PC17</td><td>CAUTION Toxic and flammable</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S3139</td><td>Make 1 M solutions of each and mix in equal amount to obtain a solution of the appropriate pH</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S3264</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888-M</td><td>5 M solution</td></tr><tr><td>Phenol, low pH.&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P4682</td><td>Store in the dark at 4 &amp;deg;C. CAUTION toxic</td></tr><tr><td>Phenol Chloroform 5:1 (125:24:1) low pH.&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1944</td><td>Store in the dark at 4 &amp;deg;C. CAUTION toxic</td></tr><tr><td>Pierce Spin Columns</td><td>Thermo Scientific</td><td>69702</td><td>Optional</td></tr><tr><td>SCSM single drop-out &amp;ndash;ura</td><td>Formedium&amp;nbsp;</td><td>DSCS101</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich</td><td>32318-M</td><td>Make a 3 M solution and pH to 5.3 with acetic acid</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>795429</td><td>CAUTION corrosive</td></tr><tr><td>SDS (Sodium dodecyl sulfate)</td><td>Sigma-Aldrich</td><td>436143</td><td>CAUTION irritant, do not inhale</td></tr><tr><td>Streptavidin Magnetic beads</td><td>NEB</td><td>1420S</td><td>Store at 4&amp;deg;C&amp;nbsp;</td></tr><tr><td>SUPERase-In, RNase inhibitor</td><td>Life technologies&amp;nbsp;</td><td>AM2696</td><td>Store at -20&amp;deg;C</td></tr><tr><td>Thiolated &lt;em&gt;Schizosaccharomyces pombe&lt;/em&gt; for spike</td><td></td><td></td><td>See section 1.7 of the protocol</td></tr><tr><td>4-thiouracil (4tU)&amp;nbsp;</td><td>ACROS ORGANICS</td><td>359930010</td><td>Store in the dark. Make 100 mM Stock in 1M NaOH, store solutions at -20&amp;deg;C.&amp;nbsp;</td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>93362</td><td>1 M solutions at various pH</td></tr><tr><td>tRNA</td><td>Sigma-Aldrich</td><td>10109541001</td><td>5mg/ml, store at -20&amp;deg;C</td></tr><tr><td>Uridine</td><td>Sigma-Aldrich</td><td>U3750</td><td>Make 1 M solution in H&lt;sub&gt;2&lt;/sub&gt;O. Split into 2 mL aliquots and store at -20 C.</td></tr><tr><td>Yeast nitrogen base without amino acids&amp;nbsp; with amonium sulphate</td><td>Formedium</td><td>CYN0410</td><td></td></tr><tr><td>Zeba Columns 0.5ml</td><td>Thermo Scientific</td><td>89882</td><td>Store at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Zirconia beads</td><td>Thistle Scientific</td><td>110791052</td><td></td></tr><tr><td>&lt;strong&gt;Equipment and Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Beadbeater</td><td>Biospec</td><td>112011EUR</td><td>Other homogenisers can be used; the correct conditions for each homogeniser and strain must be established.</td></tr><tr><td>Bioanalyser (Agilent) or similar to assess RNA quality. If this is not important a spectrophotometer is useful to quantify the RNA.</td><td></td><td></td><td></td></tr><tr><td>Centrifuge: capable of spinning cultures at 4 &amp;deg;C and at least 3000 g. Pre-chill if possible.</td><td></td><td></td><td></td></tr><tr><td>Centrifuge: capable of spinning up to 2 mL tubes at variable speeds upto 13,000 g and down to 1000 g&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Magnetic rack for separating the beads from the sample. The one used in the paper is 3D printed, available from Thingiverse (thing:3562952). Comercially available racks exist&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>PCR machine with a heated lid that will allow incubation in the dark.</td><td></td><td></td><td></td></tr><tr><td>Rotating wheel to rotate 1.5 mL tubes end over end&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Shaking heating block (such as Eppendorf Thermomixer) is recomended&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Tubes, centrifuge, Low retention, RNase free 0.5mL</td><td>Eppendorf</td><td>H179467N</td><td></td></tr><tr><td>Tubes, centrifuge, Low retention, RNase free 1.5mL</td><td>Ambion</td><td>AM12350</td><td></td></tr><tr><td>Tubes, centrifuge, 50 mL&amp;nbsp;</td><td>Sarstedt</td><td>62.547.004</td><td>Other centrifuge tubes are not gas proof allowing CO&lt;sub&gt;2&lt;/sub&gt; to disolve in the methanol, this comes out of solution vigorously on adding warm culture, leading to sample loss</td></tr><tr><td>Tubes, centrifuge, 15 mL&amp;nbsp;</td><td>Sarstedt</td><td>62.554.001</td><td></td></tr><tr><td>Tubes, 2 mL, screw cap</td><td>Greiner</td><td>723361</td><td></td></tr><tr><td>Tubes 0.2 mL strip of 8 with integral lids</td><td>Brand</td><td>781332</td><td></td></tr></tbody></table>

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.

Typical yields for nsRNA recovered using this ers4tU protocol are displayed in Figure 1b, this has been produced by a bioanalyzer and the trace shows yield of RNA versus size (nucleotides [nt]). Note, in both the bioanalyzer trace and the inset graph, that RNA recovery from time point 0 is a very small portion of that recovered from longer time points - approximately 0.3 &#181;g of RNA recovered from approximately 109 cells compared with over twice as much after just 30 s of labelling (0.8 &#181;g of nsRNA) from the same number of cells. RNA recovery at 15 s is more variable as small differences in performing the sampling have a proportionately larger effect on RNA recovery. In the bioanalyzer trace, rRNA precursors can be seen as a peak near 1000 nt and a doublet of peaks at 1700&#8722;1800 nt. The abundance of these intermediates increases as thiolation continues.
Thio-labelling was used to quantify splicing of the ACT1 transcript (Figure 3). Thiolation was performed and samples taken at 15 s intervals from the start of thio-labelling and the processing of ACT1 RNA monitored (Figure 3a,b). As can be seen, pre-mRNA is generated (by transcription), and lariats (by the first step of splicing from pre-mRNA), even after just 15 s of labelling. After about 45 s to 1 min, the amounts of lariats and pre-mRNA reach equilibrium with as much of these RNA species being created by transcription as are processed away by splicing.
To produce the data shown in Figure 3c the strain was pulsed with 4tU for 25 s and then chased with uridine. The generation of pre-mRNA and lariats reaches a maximum at 1 minute. This compares well with Figure 3b; the maximum being achieved after 45 s to reach equilibrium plus the 25 s of the labelling. After the peak, the levels decline as the thio-labelled RNAs are chased through the splicing process.
Figure 3d shows depletion of a protein splicing factor and its effect on RNA metabolism, using the &#946;-est AID 4U system6,7. Here, Prp16p is reduced from near physiological levels to 5% of this level after 25 min of depletion. Prp16p is an essential splicing factor for the second step of splicing15. Lariats are removed during the second step of splicing (Figure 3a), but here they increase above the level of pre-mRNA as Prp16 becomes limiting. At later depletion times, other factors become limiting due to secondary effects, so that levels of lariat decrease, and pre-mRNA levels rise. The level of spliced mRNA declines.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59952/59952fig1.jpg" /> 
Figure 1: Growth in 4tU and RNA recovery. (a) 4-thiouracil affects growth. Increasing the concentration of 4tU in YMM drop-out growth medium without uracil increases the doubling time of S. cerevisiae (BY4741) carrying the p4Fui&#916;PEST plasmid. Growth of four replicate cultures was monitored at 30 &#176;C in a Tecan Infinite Pro 200. All cultures were in log phase throughout, with OD600 between 0.1 and 0.6. Mock is a control culture with an equivalent amount of NaOH added, which does not by itself change the growth rate. This graph demonstrates that thio-labelling is compromise between rapid labelling and damage to the cell. Error bars are standard error of 4 replicates. (b) nsRNA yield increases linearly from about 15 s of labelling. The main figure shows the bioanalyzer traces of purified, nsRNA from 0 (not thiolated) to 2 min after addition of 4tU at 15 s intervals. Note that the 15 s sample is not shown, as it was indistinguishable from the unlabelled sample. The two large peaks correspond to ribosomal RNAs (rRNAs). The rRNA precursors and intermediates are visible as several peaks at greater molecular weight than mature rRNAs. The recovery of these precursors and intermediates increases with time. Results from one representative experiment are shown. The inset graph shows the recovery of nsRNA with increasing incubation with 4tU. The yield of nsRNA increases with increasing time of growth with 4tU. The recovery is remarkably linear (R2 = 0.934) throughout the timescale of this experiment and shows a slight increase over background even at 15 s labelling with 4tU even though not distinguishable from the unlabelled sample by eye from the bioanalyzer trace. Error bars show standard error for three biological replicates. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59952/59952fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59952/59952fig2.jpg" /> 
Figure 2: &#946;-est AID 4U &#946;-est AID 4U graphical protocol. A graphical summary of the protocol of the &#946;-est AID 4U protocol. &#946;-estradiol (&#946;-est) promotes the expression of the auxin inducible degron (AID) system which in turn depletes an AID* tagged target protein, refer to Barrass et al.7 for a detailed protocol. In this case, degron system expression is initiated 25 min before protein degradation commences and thiolation at each time point is for 1 min. Samples are taken before induction and every 2 minutes during depletion. An animated version appears in the Supplementary Figure 2. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59952/59952fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59952/59952fig3v2.jpg" /> 
Figure 3: Precursors and intermediates of ACT1 RNA splicing. Splicing of ACT1 pre-mRNA transcripts was monitored by quantitative reverse transcription PCR16. The levels of ACT1 precursor (pre-mRNA), intermediate lariat-exon2 (Lariat) and spliced product (mRNA) are shown normalized against the level of ACT1 Exon2 and steady state levels of these RNAs. (a) Location of qPCR products on the ACT1 transcript. Schematic of the locations of the qPCR products used to assay the levels of precursors, intermediates and products of the splicing reaction of the ACT1 transcripts16. Exons are represented by boxes, intron as a line and the qPCR products as lines with diamonds at either end, the color matches those used in the graphs. The pre-mRNA PCR is specific for pre-mRNA and not any intermediates of splicing as this product crosses the branch point which is disrupted after the first step of splicing. Lariat PCR will detect the product of the first step of splicing and the excised lariat produced after the second. The mRNA PCR is specific for the product of splicing, mRNA. Results from the exon PCR (present in all precursors, intermediates and products, except the excised lariat) is not shown in the graphs as this was used to normalize the data and is therefore always equal to 1. (b) Continuous thiolabelling. The amount of pre-mRNA increases with time as 4tU is incorporated by transcription and, after a short delay, splicing converts it to lariat-exon2 intermediate and spliced products. The levels of these pre-mRNA and lariat species are detectable above background after as little as 15 s of growth with 4tU and reach a maximum after approximately 45 s of continuous labelling with 4tU, at which point their production is balanced by conversion to spliced mRNA and/or degradation. Values are normalized to their steady state (left-most point of the graph), and exon 2 levels to show their appearance and processing in comparison to transcription of exon 2. As RNA splicing of ACT1 is largely co-transcriptional4,17 spliced mRNA rapidly becomes the most abundant species, its level is similar to that of exon 2. Standard error of three biological replicates, each assayed in triplicate. (c) Pulse/chase. Thiolation pulse of 25 seconds followed by chase with uridine. Compared to the steady state levels of these RNAs (left-most point), they are initially very abundant in the newly synthesized pool. The levels gradually decline as they are processed into mRNA (or degraded), approaching levels very similar to steady state levels by 5 min. Standard error of three biological replicates, each assayed in triplicate. (d) nsRNA and protein depletion. Splicing of ACT1 pre-mRNA transcripts monitored by quantitative reverse transcription PCR as in panel (a) upon depletion of the Prp16 protein using the auxin degron system as described in Figure 2. The Prp16 protein levels are also displayed in the graph plotted against the second Y-axis as percentage of levels prior to auxin depletion. Prp16 is a vital component of the spliceosome, particularly important for the second step of splicing shown in panel (a), after which lariats are degraded. When this step becomes limiting lariats accumulate initially. At later time points splicing fails completely, lariats are no longer produced and pre-mRNA levels rise. Error bars are standard error of three biological replicates, each assayed in triplicate. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59952/59952fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59952/59952fig4.jpg" /> 
Figure 4: Graphical summary of the protocol sections 1 to 3. The cells are thiolated with 4tU and allowed to grow to incorporate the modified nucleotide into the RNA. A thiolated S. pombe spike can be added to allow normalization across time points and experiments. The pulse of 4tU can be chased using un-thiolated uridine. Labelling can either be performed continuously from 4tU addition or from a change to growth conditions, the culture split and 4tU added to cultures at increasing times from the growth condition change, but each labelling only for a brief time. The cells are collected, and RNA prepared from the cells, preferably using a homogenizer and phenol-based methods. The RNA is biotinylated and then the biotinylated RNA purified from unincorporated biotin using a size exclusion column. The nsRNA is now ready for purification with streptavidin beads (section 4, Figure 5). Numbers in red correspond to the step numbers in the protocol. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59952/59952fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59952/59952fig5.jpg" /> 
Figure 5: Graphical summary of the protocol section 4. Following on from sections 1 to 3 (Figure 4), the streptavidin beads are blocked and the biotinylated RNA sample added to the prepared beads. The biotinylated RNA binds to the streptavidin beads and the un-biotinylated RNA removed and washed. The biotinylated RNA is eluted from the beads using &#946;ME and precipitated ready for further research. Numbers in red correspond to the step numbers in the protocol. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59952/59952fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Improvement of nsRNA recovery from yeast cells with and without additional copies of the importer at 1 and 3 minutes of thio-labelling. Note that Fui1 is the yeast&#39;s own promoter expressed from a 2 &#181;m plasmid. The genomic copy of this gene is present in both of these strains. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59952/Figure_S1.ai">Please click here to download this file.</a>
Supplementary Figure 2: Animated version of the &#946;-est AID 4U &#946;-est AID 4U graphical protocol. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59952/Figure_S2_Best-AID_4U.mp4">Please click here to download this file.</a>
Supplementary File 1:&#160;4tU_experiment_template.xltx.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59952/4tU_experiment_template.xltx">Please click here to download this file.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plasmid Name</td>
			<td>Importer/permease</td>
			<td>Marker</td>
			<td>Comment</td>
			<td></td>
		</tr>
		<tr>
			<td>p4Fui</td>
			<td>S. cerevisiae Fui1</td>
			<td>URA3</td>
			<td rowspan="2">Fui1 imports Uracil and Uridine, making it ideal for pulse/chase experiments.</td>
			<td></td>
		</tr>
		<tr>
			<td>pAT2</td>
			<td>S. cerevisiae Fui1</td>
			<td>LEU2</td>
			<td></td>
		</tr>
		<tr>
			<td>p4Fui-&#916;PEST</td>
			<td>S. cerevisiae Fui1</td>
			<td>URA3</td>
			<td>The PEST motif of Fui1 has been deactivated, so the permease is not degraded when there is sufficient intracellular uracil for the cell&#8217;s needs. Works well in labelling experiments and improves pulse/chase performance.</td>
			<td></td>
		</tr>
		<tr>
			<td>p4Fur</td>
			<td>S. cerevisiae Fur4</td>
			<td>URA3</td>
			<td>Uracil permease</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">YEpEBI311</td>
			<td>H. Sapiens ENT1</td>
			<td rowspan="2">LEU2</td>
			<td rowspan="2">Miller et al.11. Also contains an HSV thymidine kinase gene.</td>
			<td></td>
		</tr>
		<tr>
			<td>(equilibrative nucleoside transporter)</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">All plasmids are 2 &#181;m based. All p4 plasmids and pAT are based on the pRS16 series of plasmids. FUI1 and FUR4 are expressed from their own, endogenous promoters.</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Plasmids used with this protocol.

Watch this Scientific Journal Video about Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU at JoVE.com

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.

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 ...

extremely-rapid-specific-metabolic-labelling-rna-vivo-with-4

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

10.7K Views.  University of Edinburgh. The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

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

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

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 ...

Genetics

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as &#945;-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biology

Dual-Color End-Labeling and Vesicle Encapsulation of RNAs for smFRET-TIRF Studies

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their conformational changes over time. To monitor RNA dynamics with smFRET, we developed a method to covalently label RNAs at their termini with a FRET pair of fluorophores. This direct end-labeling strategy targets the 5&#39;-phosphate by carbodiimide (EDC)/N-hydroxysuccinimide (NHS) activation and the 3&#39;-ribose by periodate oxidation, which can be adapted to other RNAs regardless of their size and sequence to study them independently of artificial modifications. Furthermore, the 5&#39;-EDC/NHS activation is of general interest to all nucleic acids with a 5&#39;-phosphate. The use of commercially available chemicals eliminates the need to synthesize RNA-specific probes.
Total Internal Reflection Fluorescence (TIRF) microscopy requires the surface-immobilized molecules of interest to be within the evanescent field to be illuminated. A sophisticated way of keeping the RNA molecules within the evanescent field is to encapsulate them in phospholipid vesicles. Encapsulation benefits from the best of both worlds, tethering the molecule to the surface while enabling free diffusion of the molecule. We ensure that each vesicle contains only a single RNA molecule, enabling single-molecule imaging. Upon dual-end labeling and encapsulation of the RNA of interest, smFRET measurements offer a dynamic and detailed view of RNA behavior.

This article describes the dual-color labeling of long RNAs at termini positions and their surface immobilization via encapsulation in phospholipid vesicles for single-molecule FRET TIRF microscopy applications. Combining these techniques enables precise visualization and analysis of RNA dynamics at the single-molecule level.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR.
This video introduces the concepts of metabolic labeling and show two general procedures.&#160; The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos.

<div class="chapter_text" id="script_0">
Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications.
</div>
<div class="chapter_text" id="script_1">
Metabolic labeling can be conducted using a number of strategies. Here we will describe isotopic, photoreactive, and bio-orthogonal labeling.
Isotopic labeling is performed using structural analogs that are chemically identical to their natural counterparts, but have uncommon isotopes incorporated into their structure. In this L-lysine analog the carbon and nitrogen atoms are replaced with carbon-13 and nitrogen-15. Cells cultured in the presence of isotopic analogs will incorporate them into their biochemical structures. Metabolites are collected from the cells and purified for analysis. Samples with stable isotopes are analyzed using techniques such as mass spectrometry or NMR spectroscopy. Samples with radioactive labels are analyzed using liquid scintillation counting and x-ray films, which will be demonstrated in the isotopic labeling protocol.
Photoreactive labels are functional groups incorporated into proteins, which are stable until exposed to ultraviolet light. The functional group forms a reactive radical, which binds to the nearest protein. A common example, L-photo-leucine, contains a diazirine ring, which is a photoreactive crosslinker. In contrast to isotopic labeling, there is some chemical dissimilarity between photo-reactive chemical analogs and their natural counterparts. Cells may preferentially incorporate natural compounds over analogs. Therefore, it is important to perform photo-reactive labeling in medium free of the compound being mimicked. Once exposed to ultraviolet radiation, the photo-reactive groups in a labeled protein become unstable and highly reactive, causing it to cross-link with interacting proteins, creating a protein complex. Cross-linked complexes, act as snapshots that can then be analyzed using SDS-PAGE and mass spectrometry methods. This provides insights into what reactions are occurring in the metabolic pathway by identifying reaction species and how they interact by determining binding sites.
Bioorthogonal labeling strategies utilize analogs with small functional groups that have little to no reactivity with natural biomolecules. For example, azides are small functional groups, whose reactivity is said to be orthogonal to biochemical reactions. In the Staudinger ligation, a phosphine group attacks the azido group. This yields a transition state that intramolecularly reacts with a nearby ester, resulting in an amine-bonded ligand. Bioorthogonal functional groups incorporated into biomolecules can be ligated with detection tags such as fluorescent functional groups, and affinity tags such as antigens.
Now that some concepts and strategies for metabolic labeling have been discussed, let&#39;s look at the process in the laboratory.
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The first step in a metabolic labeling experiment is to collect the protein of interest. To do this, cells are grown on a plate, and an expression method is used to promote synthesis of the desired protein. In this example, leucine-rich repeat kinases, or LRRK, are expressed. Disodium phosphate, containing radioactive phosphorus-32, is used as the analog. Proper measures must be taken to protect against ionizing radiation. This includes setting up a work area, wearing proper protective equipment, and checking for radioactive contamination. Once safety measures have been taken, the medium containing the isotopic analogs is prepared. The medium from the culture is removed and, replaced with one containing the isotopic chemical analogs and then incubated. Following incubation, the cells are lysed. The lysate is collected and purified.
After purification, proteins are resolved using SDS-PAGE and then transferred to a PVDF membrane. Autoradiography is performed by exposing the membrane to x-ray film and measured using a phosphor imager. Western blotting is used to measure relative protein levels in the PVDF membrane. In this example the phosphorylation levels of leucine-rich repeat kinases synthesized in 293T cells were measured. The autoradiogram shows how much phosphorous was incorporated into the protein. Western blotting elucidates the levels of the LRRK proteins. Image analysis software is used to obtain quantitative data of phosphorylation levels of the proteins.
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In this next procedure, photoreactive labeling is demonstrated. First, the cells are prepared and cultured. The photoreactive analog is added to the cells at the mid-log phase and incubated. In this procedure p-benzoylphenylalanine is used. Samples are collected over intervals and put on ice. The samples are then exposed to obtain snapshots of the biochemical pathways over time. The proteins of interest are then purified and resolved using SDS-PAGE.
A photoreactive labeling strategy was used to identify compounds that interact with the protein of interest. Immunodetection with Western blotting shows protein bands that indicate higher molecular weight proteins are present in the irradiated samples. These are from cross-linking due to protein-protein interaction occurring during the UV irradiation.
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Now that we&#39;ve reviewed metabolic labeling procedures, let&#39;s look at some of the ways the process is used.
Metabolic labeling concepts can be extended to multicellular organisms. Plants are grown in a sealed environment, rich in stable isotopes to produced labeled plant material. Carbon dioxide containing carbon-13 is added to the enclosure, while nitrogen-15 rich fertilizer is used. The resulting harvested plant material can help answer questions about carbon and nitrogen cycling from the ecosystem.
Labeling enables the separation of newly synthesized RNA from older RNA. By changing the initial concentration of analog, the kinetics of new RNA synthesis can be determined. The results show that the concentration of 4-thiouridine affects how much new RNA is transcribed. Additionally, incorporation rates of the label into RNA can be directly quantified with a spectrophotometer.
Using biorthogonal click chemistry, glycans in a zebra fish embryo can be labeled. The eggs are injected with a labeling compound that results in alkyne labels on the glycans. The glycans in the larvae are then ligated to a dye compound at the desired development stage. The glycans in the embryos are then imaged. Glycans produced at different time points can be identified by labeling using different colors at different stages of embryo development.
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You&#39;ve just watched JoVE&#39;s video on metabolic labeling. This video described the concepts behind metabolic labeling and their strategies, went over two general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Isotopic, Photoreactive and Bioorthogonal Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs ...

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