This work was supported by the Czech Science Foundation (18-10035S), the National Sustainability Program I (LO1419), institutional support (RVO68378050), the European Regional Development Fund (CZ.02.1.01/0.0/0.0/16_013/0001775) and the Grant Agency of Charles University (GAUK 134516). We further acknowledge the Light Microscopy Core Facility, IMG CAS, Prague, Czech Republic (supported by grants (Czech-Bioimaging - LM2015062).

1. Preparation of snRNAs for Microinjection
<ol>
	<li>Prepare a DNA template containing the full-length or truncated/mutated version of snRNA by PCR using a following PCR setup: 98 &#176;C for 60 s, 98 &#176;C for 15 s, 68 &#176;C for 30 s, 72 &#176;C for 1 min, 98 &#176;C for 15 s for 35x, and 72 &#176;C for 5 min.</li>
	<li>The forward primer must contain a promoter sequence for in vitro transcription just upstream of the first transcribed nucleotide. Amplify U2 snRNA sequence using the forward primer containing the T7 promoter and the reverse primer, which ends with the last transcribed nucleotide (see Table of Materials for details).</li>
	<li>Synthesize snRNA using a kit for short RNA synthesis (see&#160;Table of Materials&#160;for details) containing T7 RNA polymerase. Add 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.8 mM UTP, 0.2 mM UTP-Alexa488, 1.8 mM trimethylguanosine cap analogue (m32,2,7G(5&#8217;)ppp(5&#8217;)G), 1 &#956;L of an RNA inhibitor and 500-700 ng of the DNA template to the reaction mixture and incubate at 37 &#730;C overnight.</li>
	<li>Add 1 &#181;L (2U) of DNase into the reaction and incubate for additional 15 min at 37 &#730;C.</li>
	<li>Isolate snRNA by acidic phenol/chloroform extraction. Remove the top water phase and add 1/10 of the original volume of 3 M sodium acetate (pH = 5.2), 3 &#181;L of glycogen for better pellet visualization and 2.5 volumes of 100% ethanol. Incubate for 1 h at -80 &#730;C, centrifuge for 10 min at 14,000 x g and 4 &#176;C.</li>
	<li>Wash the pellet with 70% ethanol and dissolve in 12 &#181;L of nuclease-free water. The usual RNA yield was around 600 ng/&#956;L. Store at -80 &#176;C.</li>
	<li>Monitor the integrity of in vitro transcribed snRNAs by agarose gel electrophoreses.</li>
	<li>Before microinjection, dilute RNA to the final concentration 200 ng/&#956;L in water containing 10 &#181;g/&#181;L dextran-TRITC 70 kDa.</li>
</ol>
2. Cells
<ol>
	<li>Before the use, treat the coverslips with 1 M HCl for 1 h, wash thoroughly in distilled water and store in 100% ethanol. Acidic treatment promotes cells adhesion to prevent cell peeling during or after injection. For easier identification of injected cells, use a coverslip with a grid.</li>
	<li>Seed HeLa or other adherent cells 1 day before microinjection on 12 mm coverslips (no. 1 or no. 1.5) to reach 50% confluency at the time of microinjection.</li>
</ol>
3. Injection
NOTE: HeLa cells were microinjected in the Dulbecco&#39;s Modified Eagle Medium (D-MEM, 4.5 g/L D-glucose containing phenol red and antibiotics). Injection was carried out using an injector and a micromanipulator equipped with the sterile needle (see Table of Materials for details). The whole microscopic/micromanipulator system was pre-heated to 37 &#176;C for at least 4 h to prevent fluctuation of individual parts of the microscope and the microinjector.
<ol>
	<li>Put the Petri dish (30 mm) containing 2 mL of culture media with the coverslip in the middle into the holder of the microscope. Load 3 &#181;L of snRNA mixture into the needle using a microloader. Install the needle into the holder of the microinjector in 45&#176; with respect to the surface of the Petri dish.</li>
	<li>To find the needle, use a 10x long distance objective. First, set the speed COARSE on the injector and lower the needle until it touches the culture medium. Using the microscope binoculars, find the bright spot, which is the place where the needle touches the culture medium.</li>
	<li>Change the speed to FINE and further lower the needle while looking into the microscope until the tip of the needle is observed. Move the needle in the middle of visual field and switch to a 40x long distance objective.</li>
	<li>After changing the objective, select the cell and move the needle above this cell. Set the pressures and time for the cell contact (see Tips and troubleshooting in discussion).</li>
	<li>Move the needle down into the cell and then set the lower limit on the micromanipulator. Be careful not to move the needle below the cell.</li>
	<li>After setting the lower limit, move the needle back above the cell and press the injection button on the joystick of the injector. The needle moves automatically inside the cell to the place where the limit is set and injects the RNA mixture.</li>
	<li>To finish, push the button MENU on the injector and remove the needle from the holder. Then disconnect the tube from the injector before switching off the injector and the micromanipulator.</li>
</ol>
4. Cell Fixation and Staining
<ol>
	<li>After the microinjection, return the cells into a CO2 incubator and incubate at 37 &#176;C for 1 h.</li>
	<li>After the incubation, rinse the cells three times with phosphate buffered saline solution (PBS) at room temperature, fix for 20 min with 4% paraformaldehyde in 0.1 M PIPES pH 6.9, and wash three times with room temperature PBS. If only snRNA localization is analyzed, briefly rinse the cells in water and mount in a mounting medium with DAPI.</li>
	<li>In case of additional protein localization, permeabilize the cells with 0.5% Triton-X100 in PBS for 5 min at room temperature. After three rinses with PBS, incubate the cells with primary and secondary antibodies and after final washes in PBS and brief rinse in water, mount in a mounting medium with DAPI.</li>
</ol>
5. Microscopy
<ol>
	<li>If not imaged immediately after mounting, store the slides at 4 &#176;C until imaging.</li>
	<li>Acquire images using a high-end fluorescence microscopic system equipped with an immersion objective (60X or 100x/1.4NA).</li>
	<li>Once microinjected cells are identified, collect a stack of 20 z-sections with 200 nm z steps per sample and subject to mathematical deconvolution. Maximal projections or individual z stacks were then presented.</li>
	<li>To quantify the fluorescent signal, draw Region of Interest (ROI) around a Cajal body identified by coilin immunostaining. Measure the intensity of the snRNA signal in the coilin-defined ROI. Next determine the intensity of snRNA fluorescence in ROI randomly placed in the nucleoplasm and calculate the ratio of RNA signal in the Cajal body and nucleoplasm.</li>
</ol>

<ol><li>Ishikawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+truncated+forms+of+U1+snRNA+reveals+a+novel+RNA+degradation+pathway+during+snRNP+biogenesis%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Identification of truncated forms of U1 snRNA reveals a novel RNA degradation pathway during snRNP biogenesis.</a> Nucleic Acids Research. 42 (4), 2708-2724 (2014).</li>
<li>Stanek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cajal+bodies+and+snRNPs+-+friends+with+benefits%5BTitle%5D)+AND+%22RNA+Biology%22%5BJournal%5D)">Cajal bodies and snRNPs - friends with benefits.</a> RNA Biology. 14 (6), 671-679 (2017).</li>
<li>Stanek, D., Fox, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+bodies%3A+news+insights+into+structure+and+function%5BTitle%5D)+AND+%22Curentr+Opinion+in+Cell+Biology%22%5BJournal%5D)">Nuclear bodies: news insights into structure and function.</a> Curentr Opinion in Cell Biology. 46, 94-101 (2017).</li>
<li>Novotny, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SART3-Dependent+Accumulation+of+Incomplete+Spliceosomal+snRNPs+in+Cajal+Bodies%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">SART3-Dependent Accumulation of Incomplete Spliceosomal snRNPs in Cajal Bodies.</a> Cell Reports. 10, 429-440 (2015).</li>
<li>Roithova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Sm-core+mediates+the+retention+of+partially-assembled+spliceosomal+snRNPs+in+Cajal+bodies+until+their+full+maturation%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">The Sm-core mediates the retention of partially-assembled spliceosomal snRNPs in Cajal bodies until their full maturation.</a> Nucleic Acids Research. 46 (7), 3774-3790 (2018).</li>
<li>Fischer, U., Sumpter, V., Sekine, M., Satoh, T., Luhrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nucleo-cytoplasmic+transport+of+U+snRNPs%3A+definition+of+a+nuclear+location+signal+in+the+Sm+core+domain+that+binds+a+transport+receptor+independently+of+the+m3G+cap%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap.</a> EMBO Journal. 12 (2), 573-583 (1993).</li>
<li>Shukla, S., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quality+control+of+assembly-defective+U1+snRNAs+by+decapping+and+5%27-to-3%27+exonucleolytic+digestion%5BTitle%5D)+AND+%22Proceeding+of+the+National+Academy+of+U+S+A%22%5BJournal%5D)">Quality control of assembly-defective U1 snRNAs by decapping and 5'-to-3' exonucleolytic digestion.</a> Proceeding of the National Academy of U S A. 111 (32), E3277-E3286 (2014).</li>
<li>Weil, T. T., Parton, R. M., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+the+message+clear%3A+visualizing+mRNA+localization%5BTitle%5D)+AND+%22Trends+in+Cell+Biology%22%5BJournal%5D)">Making the message clear: visualizing mRNA localization.</a> Trends in Cell Biology. 20 (7), 380-390 (2010).</li>
<li>Klingauf, M., Stanek, D., Neugebauer, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancement+of+U4%2FU6+small+nuclear+ribonucleoprotein+particle+association+in+Cajal+bodies+predicted+by+mathematical+modeling%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">Enhancement of U4/U6 small nuclear ribonucleoprotein particle association in Cajal bodies predicted by mathematical modeling.</a> Molecular Biology of the Cell. 17 (12), 4972-4981 (2006).</li>
<li>Mhlanga, M. M., Vargas, D. Y., Fung, C. W., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((tRNA-linked+molecular+beacons+for+imaging+mRNAs+in+the+cytoplasm+of+living+cells%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells.</a> Nucleic Acids Research. 33 (6), 1902-1912 (2005).</li>
</ol>

The authors have nothing to disclose.

We employed microinjection of fluorescently labelled snRNAs to determine sequences important for snRNA localization into nuclear Cajal bodies. Due to rapid and rather simple preparation of labelled RNAs (preparation of DNA template by PCR followed by in vitro transcription) the method offers effective analysis of how various sequences contribute to RNA localization. In relatively short time, we were able to analyze ten different deletions or substitutions of the U2 snRNA (reference5 and data not shown). For RNAs with a complex biogenesis pathway, this approach also allows testing sequences that are essential for maturation. In the case of snRNAs, the deletion of Sm binding sequences, which are required for Sm ring assembly results in sequestration and degradation of mutated snRNAs in the cytoplasm7. Other advantageous include that two differently labelled RNAs can be injected simultaneously and their localization directly compared within one cell. However, as various fluorochromes might modify RNA behavior, each fluorochrome must be tested individual before double labelling experiments. RNAs of several hundred nucleotides in length have been successfully injected and their localization assayed in various model systems (reviewed in reference8). The limiting step in case of longer RNAs is usually in vitro synthesis of full-length RNA. Also, injection of RNAs with pre-assembled proteins can be applied to test the effect of individual proteins on RNP localization. In our projects, we injected snRNA associated with Sm proteins to confirm the role of the Sm ring in Cajal body localization5. Finally, microinjection of fluorescently labelled RNAs allows direct quantification without any additional steps, as has been previously applied for snRNAs9.
There are also several disadvantages of the methods that should be considered before launching a microinjection project. First, despite some automatization of the microinjection process, the method still requires manual skills and experience, and researchers should consider time necessary for training. The critical parts of the protocol are the preparation of intact RNAs, microinjection and then finding injected cells in the microscope (see Tips and Troubleshooting below). Second, microinjection represents a significant stress and many cells do not survive for a longer period of time. While there are some successful attempts to follow microinjected RNAs inside cells by live-cell imaging10, we observed significant increase of cell death when we combined microinjection with live-cell imaging using fluorescence microscopy. Third, the amount of injected RNA into human cells is very low which prevents biochemical analysis of injected RNAs. This also means that it is difficult to monitor how the incorporation of fluorescently labelled nucleotides affects RNA functions. Therefore, various ratios of labelled-NTP:NTP should be incorporated into wild-type RNA and its localization assayed to get the ideal ratio between the fluorescence signal and the functionality of labelled RNA. Also, the nature of fluorochrome might affect RNA localization. In our hands, Alexa488-UTP worked much better than Alexa546-UTP. We have not explored these differences any further, but one reason could be a bigger size and different shape of Alexa546 in comparison to Alexa488.
Taking together, RNA microinjection is a powerful method for RNA localization but should be always combined with alternative approaches. In our case, we successfully introduced MS2-binding site into U2 snRNA, monitored its localization using MS2-YFP and confirmed some key results obtained by snRNA microinjection with the MS2 system5.
Tips and troubleshooting 
Injection pressure, compensation pressure and injection time need to be determined experimentally for each cell line. We applied injection pressure (Pi) 170 hPa and compensation pressure (Pc) 50 hPa in the case of cytoplasmic microinjection and Pi=200 hPa and Pc=80 hPa in the case of nuclear microinjection. The injection time was in both cases set to 0.2 s. You can sometimes observe a slight movement of material inside the cell, which is an indication of a successful injection. If the cell bursts, you have to decrease the injection pressure. You should also check the compensation pressure via the TRITC fluorescence channel. When you see the strong stream coming out from the tip of the needle, then decrease the compensation pressure.
Between injections, always check whether the cells are successfully injected. Identify the microinjected cells via injected Dextran-TRITC using the TRITC fluorescence channel. If you do not see any microinjected cells, first increase the injection pressure and injection time. Second, check if the needle is not plugged via fluorescence channel (TRITC). If you see Dextran-TRITC weakly streaming from the tip of the needle, then the needle is functional, and the problem is likely in the setting of the lower limit for injection. The setting of the limit is a very tricky and important step. Each cell has different shape and you will to change the limit very often to ensure proper microinjection.
In the ideal case, one should be able to microinject ~50 cells before the tip of the needle is plugged with a cell debris. Check if fluorescence comes out of the tip regularly, and if you do not see any Dextran-TRITC streaming from the tip, then the needle is blocked. To clean it, hold the CLEAN button on the injector for 3 s, which increases pressure and should remove the block. If it does not help, then you could try to break off the tip of the needle by hitting the bottom of the Petri dish. However, this is a very tricky procedure, which requires a significant amount of experience and there is no guarantee of success. Therefore, for beginners, the needle exchange is advisable.
Before you exchange the needle, push the MENU button on the injector. Take the needle a little bit up and press HOME on the micromanipulator. The needle will go all the way up from the medium, but the micromanipulator remembers the original position of the needle and you do not need to find the needle again. When the needle is replaced, press the HOME button and the needle will go to the original position and you should be able to see the tip of the needle in the microscope. After changing the needle, you have to adjust the limit. Then, push the MENU button on the injector again and the needle is prepared for microinjection.
During imaging, the trickiest part is to find the microinjected cells. To locate them, use TRITC channel, where the TRITC-conjugated Dextran-70kDa is much better visible than a weak Alexa488 signal of microinjected snRNAs. The coverslips with a grid are helpful here.

RNA splicing is one of the crucial steps in gene expression, which is catalyzed by a large ribonucleoprotein complex called the spliceosome. In total, more than 150 proteins and 5 small nuclear RNAs (snRNAs) are integrated into the spliceosome at different stages of the splicing pathway. U1, U2, U4, U5 and U6 snRNAs are participating in splicing of major GU-AG introns. These snRNAs join the spliceosome as pre-formed small nuclear ribonucleoprotein particles (snRNPs) that contain snRNA, seven Sm proteins associated with snRNA (or Like-Sm proteins, which associate with the U6 snRNA) and 1-12 proteins specific for each snRNP.
Assembly of snRNPs involves cytoplasmic and nuclear stages. Newly transcribed snRNA is exported to the cytoplasm where it acquires a ring assembled from seven Sm proteins. The Sm ring subsequently serves as a signal for snRNA re-import back to the nucleus. Defective snRNAs that fail to associate with Sm proteins are retained in the cytoplasm1. Newly imported snRNPs first appear in the Cajal body where they meet snRNP-specific proteins and finish their maturation (reviewed in reference2,3). We recently showed that inhibition of final maturation steps results in sequestration of immature snRNPs in Cajal bodies4,5. We proposed a model where the final snRNP maturation is under quality control that monitors addition of snRNP-specific proteins and the formation of active snRNPs. However, molecular details of how cells distinguish between correctly assembled mature and aberrant immature particles remain elusive.
To determine snRNA sequences that are essential for targeting and accumulation of snRNAs in nuclear Cajal bodies, we decided to employ microinjection of fluorescently labelled snRNAs. Microinjection was a method of choice because: 1) it does not require an additional sequence tag to distinguish synthetic snRNAs form their endogenous counterparts which is especially important for short RNAs with little space for insertion of extra tag sequence; 2) it allows analysis of sequences that are important for biogenesis. For example, the Sm sequence is essential for Sm ring assembly and re-import into the nucleus6. When snRNAs are expressed in the cell, snRNAs lacking the Sm sequence are degraded in the cytoplasm and do not reach the nucleus and Cajal bodies7. However, snRNAs without the Sm sequence can be directly microinjected into the nucleus and thus a potential role of the Sm sequence in Cajal body localization assayed.
Here, we describe in detail a microinjection method that we applied to determine snRNA sequences necessary to target snRNAs into the Cajal body5. We showed that Sm and SMN binding sites are together necessary and sufficient to localize not only snRNAs but various short non-coding RNAs into the Cajal body. Based on microinjection as well as other evidence, we proposed that the Sm ring assembled on the Sm binding site is the Cajal body localization signal.

<table><tbody><tr><td>ChromaTide Alexa fluor 488-5-UTP</td><td>ThermoFisher</td><td>C11403</td><td>Stock concentration 1 mM</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium - high glucose</td><td>Sigma-Aldrich</td><td>D5796</td><td>Containing 4.5 g&amp;frasl;L D-glucose, Phenol red and antibiotics</td></tr><tr><td>FemtoJet express Injector</td><td>Eppendorf</td><td>5247000013</td><td></td></tr><tr><td>Femtotips II</td><td>Eppendorf</td><td>930000043</td><td>Microinjection needle of 0.5 &amp;micro;m inner and 0.7 &amp;micro;m outer diameter</td></tr><tr><td>Fluoromont G with DAPI</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>Glycogen</td><td>ThermoFisher</td><td>AM9510</td><td>Stock concentration 5 mg/mL</td></tr><tr><td>Gridded Glass Coverslips</td><td>Ibidi</td><td>10817</td><td>Coverslips with a grid, no direct experience with them</td></tr><tr><td>InjectMan NI 2 Micromanipulator</td><td>Eppendorf</td><td>5181000017</td><td></td></tr><tr><td>m3-2,2,7G(5&#039;)ppp(5&#039;)G trimethyled cap analogue</td><td>Jena Bioscience</td><td>NU-853-1</td><td>Stock concentration 40 mM</td></tr><tr><td>MEGAshortscript T7 Transcription Kit</td><td>ThermoFisher</td><td>AM1354</td><td></td></tr><tr><td>Microscope Cover Glasses 12 mm, No. 1</td><td>Paul Marienfeld GmbH</td><td>111520</td><td>For routine work</td></tr><tr><td>Microscope Cover Glasses 12 mm, No. 1.5</td><td>Paul Marienfeld GmbH</td><td>117520</td><td>For high resolution images</td></tr><tr><td>Microscope DeltaVision</td><td>GE Healthcare</td><td></td><td>For image acquisition</td></tr><tr><td>Microscope DMI6000</td><td>Leica</td><td></td><td>For microinjection</td></tr><tr><td>Paraformaldehyde 32% solution EM grade</td><td>EMS</td><td>15714</td><td>Dissolved in PIPES to the final concentration 4%</td></tr><tr><td>Phenol:Chloroform 5:1</td><td>Sigma-Aldrich</td><td>P1944</td><td></td></tr><tr><td>Primers for U2 amplification: Forward: 5&amp;rsquo;-TAATACGACT&lt;br /&gt;CACTATAGGGATCGCTTCT&lt;br /&gt;CGGCCTTTTGG,&lt;br/&gt; Reverse: 5&amp;acute; TGGTG&lt;br /&gt;CACCGTTCCTGGAGGT</td><td>Sigma-Aldrich</td><td></td><td>T7 rpromoter sequence in italics</td></tr><tr><td>Phusion High Fidelity DNA polymerase</td><td>BioLab</td><td>M0530L</td><td></td></tr><tr><td>RNasin Plus</td><td>Promega</td><td>N2615</td><td>Stock concentration 40 mM</td></tr><tr><td>Tetramethylrhodamine isothiocyanate Dextran 65-85 kDa</td><td>Sigma-Aldrich</td><td>T1162</td><td>Dissolved in water, stock concentration 1 mg/mL</td></tr><tr><td>Triton-X100</td><td>Serva</td><td>37240</td><td>Dissolved in water, stock concentration 10%</td></tr></tbody></table>

analysis of spliceosomal snrna localization in human hela cells using microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment called the Cajal body. However, sequences that direct snRNA localization into this subnuclear structure have not been known until recently. To determine sequences important for accumulation of snRNAs in Cajal bodies, we employed microinjection of fluorescently labelled snRNAs followed by their localization inside cells. First, we prepared snRNA deletion mutants, synthesized DNA templates for in vitro transcription and transcribed snRNAs in the presence of UTP coupled with Alexa488. Labelled snRNAs were mixed with 70 kDa-Dextran conjugated with TRITC, and microinjected to the nucleus or the cytoplasm of human HeLa cells. Cells were incubated for 1 h and fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence, while snRNAs and dextran, which serves as a marker of nuclear or cytoplasmic injection, were observed directly using a fluorescence microscope. This method allows for efficient and rapid testing of how various sequences influence RNA localization inside cells. Here, we show the importance of the Sm-binding sequence for efficient localization of snRNAs into the Cajal body.

To monitor snRNA localization and the role of the Sm binding site in Cajal body targeting, we prepared a DNA template containing the T7 promoter and either the full-length U2 snRNA or U2 snRNA lacking the seven nucleotides (AUUUUUG) forming the Sm binding site. snRNAs were in vitro transcribed, isolated and mixed with TRITC-coupled dextran-70kDa. We microinjected the mixture containing in vitro transcribed snRNA into the nucleus or the cytoplasm of HeLa cells.
It has been previously shown that the Sm binding site is necessary for nuclear import6. To test whether the Sm site is also important for Cajal body accumulation, we microinjected snRNA lacking the Sm site directly into the nucleus (Figure 1A). Injection into the cytoplasm served as a control (Figure 1B). As a positive control, we microinjected full-length snRNA (Figure 1C,D). After 60 min of incubation in a CO2 incubator, microinjected cells were fixed and the Cajal body marker coilin was visualized by indirect immunofluorescence. The full-length snRNA accumulated in Cajal bodies after both nuclear and cytoplasmic injections. In contrast, snRNA lacking the Sm site remained in the cytoplasm after microinjection into this compartment, which is consistent with previous findings6. The nuclear microinjection of snRNA without the Sm site revealed that the Sm binding sequence is important for Cajal body localization. U2 snRNA lacking the Sm site remained in the nucleus but did not accumulate in Cajal bodies (Figure 1A).
Sometimes, the microinjection into only one cellular compartment failed and TRICT-dextran-70kDa was found in both the nucleus and the cytoplasm (Figure 2A). These cells were discarded from further analysis. Despite the fact that the fluorescently labelled snRNAs are stored at -80 &#176;C before the injection, RNA degradation can occur. Degraded snRNA injected into the cytoplasm is not transported into the nucleus and stays localized in the cytoplasm (Figure 2B). Such snRNA should be discarded, and new snRNA should be synthesized.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59797/59797fig1.jpg" /> 
Figure 1: Localization of microinjected snRNAs. Alexa488-labelled U2 snRNA either lacking the Sm site (A,B) or full-length (C,D) were microinjected into the cytoplasm or the nucleus of HeLa cells. Cajal bodies (arrows) are marked by coilin immunolabeling (red); snRNAs are depicted in green. Dextran-TRITC-70kDa (yellow) was used to monitor nuclear or cytoplasmic injection; DNA was stained by DAPI (blue). Arrowheads marks cytoplasmic localization of snRNA. Scale bar = 10 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59797/59797fig1large.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/59797/59797fig2.jpg" /> 
Figure 2: A potential pitfall of the microinjection approach. (A) Microinjection into one compartment failed and WT U2 snRNA was microinjected into both the cytoplasm and the nucleus. Note that Dextran-TRITC-70kDa (yellow) is present in both cellular compartments. (B) WT U2 snRNA was microinjected into the cytoplasm of HeLa cells (green) but significant amount of snRNA remained in the cytoplasm (arrowheads), which indicates partial degradation of injected snRNA and a loss of the Sm binding site. Cajal bodies (arrows) are marked by coilin immunolabeling (red). DNA was stained by DAPI (blue). Scale bar = 10 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59797/59797fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection at JoVE.com

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Biogenesis of spliceosomal snRNAs is a complex process involving various cellular compartments. Here, we employed microinjection of fluorescently labelled snRNAs in order to monitor their transport inside the cell.

Biogenesis of spliceosomal snRNAs is a complex process involving both nuclear and cytoplasmic phases and the last step occurs in a nuclear compartment ...

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Nanyang Technological University

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JoVE Journal

Neuroscience

6.0K Views.  Institute of Molecular Genetics of the Czech Academy of Sciences. Hier hebben we micro-injectie van fluorescerende RNAs toegepast om RNA-sequenties te detecteren die essentieel zijn voor de lokalisatie van korte nucleaire RNAs in nucleaire structuren genaamd Cajal-lichamen. Deze techniek heeft twee grote voordelen. Ten eerste kan het worden toegepast voor korte RNAs die anders moeilijk te labelen en te volgen zijn.En ten tweede, het maakt een snelle screening van verschillende sequenties en het testen van hun rol in RNA lokalisatie. Een dag voor micr...

Video: Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Genetics

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), ...

FISH for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

Author Spotlight: RNA FISH for Locating lncRNA-SNHG6 in Osteosarcoma Cells 

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition ...

Cancer Research

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Biochemistry

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

Developmental Biology

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

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

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

Bioengineering

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 ...

Analysis of Spliceosomal snRNA Localization in Human Hela Cells Using Microinjection

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