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Sep 2021
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Labelling of Active Transcription Sites with Argonaute NRDE-3—Image Active Transcription Sites in vivo in Caenorhabditis elegans
用Argonaute NRDE-3标记活性转录位点—图像秀丽隐杆线虫体内的活性转录位点   

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Abstract

Live labelling of active transcription sites is critical to our understanding of transcriptional dynamics. In the most widely used method, RNA sequence MS2 repeats are added to the transcript of interest, on which fluorescently tagged Major Coat Protein binds, and labels transcription sites and transcripts. Here we describe another strategy, using the Argonaute protein NRDE-3, repurposed as an RNA-programmable RNA binding protein. We label active transcription sites in C. elegans embryos and larvae, without editing the gene of interest. NRDE-3 is programmed by feeding nematodes with double-stranded RNA matching the target gene. This method does not require genome editing and is inexpensive and fast to apply to many different genes.


Graphical abstract:



Keywords: Transcriptional labelling (转录标记), mRNA labelling (mRNA标记), C. elegans (秀丽隐杆线虫), Argonaute (船蛸属), In vivo imaging (活体成像), Fluorescence microscopy (荧光显微镜)

Background

We describe here a protocol to image transcription at transcription sites in C. elegans using an Argonaute protein NRDE-3, based on a method we recently published (Toudji-Zouaz et al., 2021). In C. elegans, double-stranded RNA targeting the gene of interest is uptaken by the systemic RNAi mechanisms and distributed to all cells by the dsRNA transporter SID-1.


The exoRNAi pathway will process dsRNA and use them for recognition of target mRNAs for degradation. This recruits the RNA dependent RNA polymerase RRF-1 to the transcript, which will synthesise triphosphorylated, antisense, secondary small RNAs that are loaded into secondary Argonautes. The only somatic secondary Argonaute, NRDE-3, when unladen, is cytoplasmic. Once loaded with small RNA, NRDE-3 moves to the nucleus and binds to the nascent transcript, where it recruits other proteins to silence transcription.


In a wild-type background, the endogenous RNAi pathway will induce synthesis of secondary small RNAs, and constitutive nuclear localisation of NRDE-3 (Figure 1A, B). To prevent this, we introduce a mutation in eri-1. NRDE-3 is therefore unladen in most tissues (except for the germline, intestine, and early embryo), localises to the cytoplasm, and will move into the nucleus only if the gene of interest is expressed in the cell (Guang et al., 2008) (Figure 1C, D). To prevent downstream transcriptional silencing, we also introduce a mutation in nrde-2 (Guang et al., 2010).



Figure 1. NRDE-3 nuclear localisation is dependent on small RNAs.

Nuclear localisation of fluorescently labelled NRDE-3 in the embryo (A) and adult somatic tissues (head) (B) in a eri-1(+) genetic background. Cytoplasmic localisation of NRDE-3 in a eri-1(-) background, in the embryo (C) and adult (D). Note the nuclear localisation in the germline primordium in the embryo (C).

Materials and Reagents

  1. Petri dishes, 60 mm diameter (Falcon, catalog number: 353004)

  2. Microscopy slides (Ghäasel, catalog number: 29-201-307)

  3. Coverslips (Knittel, catalog number: 100037)

  4. Sodium chloride (Roth, CAS: 7647-14-5)

  5. Bacto agar (BD, CAS: 9002-18-0)

  6. Bacto peptone (BD, CAS: 51142-18-8)

  7. 5 mg/mL cholesterol in ethanol, filter sterilised (Sigma, CAS: 57-88-5)

  8. KH2PO4 (Roth, CAS 7778-77-0)

  9. K2HPO4 (VWR, CAS: 16788-57-1)

  10. 1 M MgSO4 (Roth, CAS: 7487-88-9)

  11. 1 M CaCl2 (VWR, CAS: 10035-04-8)

  12. Ampicillin 1,000× (Sigma, CAS: 69-53-4) or carbenicillin 1,000× (Roth, CAS: 4697-36-3) 25 mg/mL (store at -20°C)

  13. IPTG (Sigma, CAS: 367-93-1), 1 M (store at -20°C)

  14. Na2HPO4 (VWR, CAS: 7558-79-4)

  15. Feeding RNAi clones targeting the gene of interest, either from libraries (Ahringer library, Source Bioscience; Vidal library, Horizon discovery, catalog number: RCE1181; stored at -80°C) or homemade

  16. Serotonin (Sigma, catalog number: H7752-5G), stock solution 25 mM in M9, keep at -20°C

  17. 0.1 µm diameter polystyrene microspheres (Polysciences, catalog number: 00876-15, 2.5% w/v suspension in M9)

  18. Nematode strains from Caenorhabditis Genetics Center (University of Minnesota): VBS662, VBS663, VBS664, VBS668

  19. Wizard Plus SV miniprep kit (Promega, catalog number: A1460)

  20. LB agar plates with 50 µg/mL ampicillin and 10 µg/mL tetracycline

  21. Liquid LB with 50 µg/mL ampicillin

  22. 1 M KPO4 buffer (see Recipes)

  23. M9 buffer (see Recipes)

Equipment

  1. Peristaltic pump (Wheaton Omnispense plus)

  2. Confocal microscope, Spinning disk Roper on Nikon Eclipse Ti, with argon laser for illumination at 515 nm

  3. Incubator (Pol Eko apartura), set at 20°C

  4. Centrifuge (Eppendorf 5804R)

  5. Worm pick, prepared according to (Stiernagle, 2006)

Software

  1. Fiji (https://fiji.sc/)

Procedure

This procedure is relatively simple and can be implemented in a few days (Figure 2). It includes preparation of RNAi plates for bacteria expressing dsRNA, exposure of nematodes, and imaging.



Figure 2. Procedure flowchart.

Preparing plates

Prepare plates with standard Nematode Growth Media (Stiernagle, 2006), supplemented with ampicillin or carbenicillin (to select bacteria carrying the plasmid of interest) and IPTG (to induce transcription of double-stranded RNA). Follow the standard guidelines for RNAi by feeding (Ahringer, 2006). Incorporating antibiotics during plate preparation, rather than afterwards, increases dsRNA expression consistency.


  1. Preparation of NGM RNAi plates

    1. Mix 3 g of NaCl, 17 g of agar, and 2.5 g of peptone in a bottle; add 975 mL of ddH2O. Autoclave for 50 min.

    2. Let bottle cool down to ~55°C.

    3. Add 1 mL of 1 M CaCl2, 1 mL of 5 mg/mL cholesterol in ethanol, 1 mL of 1 M MgSO4, 25 mL of 1 M KPO4 buffer, 1 mL of IPTG, and 1 mL of carbenicillin or ampicillin.

    4. Pour plates with the peristaltic pump, under sterile conditions next to a flame (7 mL for 60 mm diameter plates).

    5. Plates should be left to dry for at least 24 h in the dark before seeding; if too wet, the bacteria will not dry properly after seeding. For best practice, plates should be used within two weeks, although we have observed labelling with month-old plates.


    Choosing RNAi clones

    Two large libraries of RNAi clones in the E. coli strain HT115 bacteria are available: the Ahringer library (Kamath et al., 2003), no longer commercially distributed by Source Bioscience, but widely available, covering genomic sequences with ~1 kb long clones; and the Vidal ORFeome library (Rual et al., 2004), still commercially distributed by Horizon Discovery, containing full reading frames. If your gene of interest is not available in these libraries, make your own vector by cloning the ORF of interest in the L4440 empty vector (Ahringer, 2006).

    Labelling of transcription sites relies on the tiling of fluorescently tagged NRDE-3 on transcripts; it derives that, the longer the dsRNA fed to the worms, the more fluorescent proteins will accumulate on each transcript. When we tested RNAi clones targeting only one exon of hlh-1, we were able to observe nuclear localisation of NRDE-3 in muscle cells, but failed to observe active transcription sites. To maximise signal, it is therefore preferable to use long RNAi clones. The RdRP RRF-1 processes the target RNA 3’→5’; therefore, a RNAi clone in 3’ of the target transcript will induce secondary siRNAs covering more of the transcript.


  2. Preparation of RNAi cultures

    1. Streak clones from the frozen library onto LB plates containing 50 µg/mL ampicillin (to select for presence of the dsRNA coding plasmid), and 10 µg/mL tetracycline (to select for a mutation in a dsRNAse in HT115), using a sterile pipette tip or inoculation loop, and grow overnight at 37°C.

    2. (Optional) Miniprep and sequence the clone. A minority of clones in these libraries are erroneously annotated; it is therefore good practice to confirm their identity.

    3. Grow RNAi clone in 5 mL of liquid LB with 50 µg/mL ampicillin, between 8 h and overnight at 37°C and 200 rpm.

    4. Pellet down the culture, pour out the supernatant, resuspend the pellet in 200–300 µL and seed 100 µL on plates. IPTG should induce transcription of dsRNA within a few hours. We typically transfer worms on plates the next day or when plates are sufficiently dry.

    Despite the mutation in nrde-2 abrogating secondary silencing at the transcriptional level (Guang et al., 2010; Toudji-Zouaz et al., 2021), with some dsRNA clones (e.g., ant-1.1, ama-1), we observed deleterious effects (sterility, lethality), possibly due to the primary RNAi pathway being sufficient for a significant level of knockdown. By diluting the RNAi culture 1/2 to 1/5 with bacteria carrying the empty vector L4440, it is possible to bypass these deleterious effects.


    Choosing which strain to use

    Four strains are available from the CGC; all transgenes were integrated as single copy by CRISPR, at loci ttTi5605 (chromosome II) or ttTi4348 (chromosome I).


    VBS662 eri-1(mg366) IV; nrde-2(gg95) peef-1A.1::YFP::nrde-3 II

    In the absence of RNAi treatment, the YFP signal is nicely homogenous in the cytoplasm at all stages; expression under the control of the strong eef-1A.1 promoter is high; therefore, the background signal in the nucleus will be strong, and weak transcription signals will be difficult to observe. YFP also has a low photostability, making it less appropriate for timelapse imaging.


    VBS663 eri-1(mg366) IV; nrde-2(gg95) prps-27::mNeonGreen::flag::nrde-3 II

    mNeonGreen tends to form aggregates in the cytoplasm of embryos, and is therefore less optimal for imaging at this stage. In larval stages, signal is nicely homogenous. The expression level under the control of the weaker rps-27 promoter ensures a higher signal-to-noise ratio. The higher photostability makes it better for long timelapses.


    VBS668 eri-1(mg366) IV; nrde-2(gg95) peef-1A.1::YFP::nrde-3::SL2::sid-1 II

    By expressing the dsRNA transporter sid-1 in all tissues, this strain improves efficiency in neurons (Calixto et al., 2010). However, it does not reach the efficiency of other somatic tissues; several components of the RNAi pathway appear to be sparsely expressed in neurons (Cao et al., 2017). In worms fed on dsRNA targeting GFP, we observe nuclear localisation in neurons in adults, but this is less efficient in larvae. The lower efficiency means that one has to look at multiple individuals to see proper nuclear localisation and transcriptional labelling.


    VBS664 eri-1(mg366) IV; nrde-2(gg95) peef-1A.1::VenusC::nrde-3 II; peef-1A.1::VenusN::nrde-3 I
    Bipartite Venus:NRDE-3 allows reconstitution of fluorescence when multiple NRDE-3 accumulate on the same transcript; background fluorescence in nucleus and cytoplasm is therefore reduced. In those cells expressing the gene of interest, in addition to active transcription sites, the nucleoplasm is still visible: once Venus is reconstituted, it does not dissociate again; after unloading from the transcript, NRDE-3 will stay or translocate back to the nucleus, leading to some background nucleoplasm signal. Some spontaneous reconstitution is observed associated to the cytoskeleton in some cells. The reduced background allows observation of cytoplasmic transcripts as faint spots that bleach rapidly (Figure 3). Occasionally, more than two nuclear spots are observed; they might indicate transcripts in processing and export.



    Figure 3. Labelling of cytoplasmic transcripts with bipartite Venus.

    Labelling of hlh-1 transcription sites (white arrowheads) and cytoplasmic transcripts (red arrowheads) with split Venus complementation on transcripts.


  3. Exposure to dsRNA and imaging

    1. Transfer well-fed L4 hermaphrodites to RNAi plates. To reduce bacteria contamination, first pick the worms to an unseeded plate, and then transfer them to the RNAi plate. We recommend to transfer ~20 hermaphrodites.

    2. Leave worms on the plate for at least 24 h, and up to 4 days, at 20°C. Optimal exposure can vary depending on the gene and the developmental stage of interest; try out a few exposure times.

    3. For embryos, dissect gravid hermaphrodites and mount embryos, on a 2–5% agar pad in water on a slide, taking care to limit bacteria transfer as much as possible; add a coverslip and seal the edges to prevent evaporation (Walston and Hardin, 2010).

    4. For larvae and adults, mount on a 5% agar pad, with 1 to 2 µL polystyrene beads, and 2 µL of serotonin in M9 for immobilisation (Lee et al., 2019), taking care to avoid bacteria transfer; add a coverslip and seal the edges to prevent evaporation.

    5. Image on confocal microscope, preferably a spinning disk confocal microscope to reduce photobleaching. YFP, mNeonGreen, and Venus, the fluorescent proteins used in these strains, are compatible with excitation at 515 nm. Laser intensity and exposure should be adjusted to limit photobleaching; we try to stay around 0.3 mW. We perform image acquisition with a 60×/1.20 NA objective, which gives sufficient resolution and a long enough working distance, and a Z step of 0.5 µm.

    6. You should observe nuclear localisation in the cells expressing the gene of interest, and in some of these cells, one or two dots, corresponding to active transcription sites. If working with a gene with multiple paralogs, or in a polyploid tissue (e g., gut or hypodermis), more than two dots can be observed. The transcription size spots should have a diameter <1 µm; the size should be diffraction limited. Theoretically, spots should become observable as soon as NRDE-3 moves to the nucleus.


    After exposure to dsRNA over multiple generations, transcription sites are less often observed. We therefore recommend limiting exposure to one or two generations.

      Alternative immobilisation methods are discouraged: sodium azide, being a general metabolic poison, is best avoided; while transcription spots can be observed, we have generally failed to observe dynamics with azide. Levamisole can work, provided concentrations are low enough.

    While an epifluorescence microscope is sufficient to observe nuclear localisation, it is unlikely to successfully image transcription spots. A confocal microscope is necessary to successfully differentiate the weak transcription site from the background. Imaging over multiple hours is taxing on the worms, due to phototoxicity and oxygen deprivation; with mNeonGreen::NRDE-3, after multiple hours, non-specific spots can appear in the cytoplasm. It is therefore preferable to limit laser power (<0.4 mW) and limit the amount of bacteria transferred to the slide.


    Positive and negative controls

    As a positive control, we recommend targeting the gene hlh-1, coding for the MyoD orthologue, expressed in all muscle cells at a moderate level. As a negative control, we use the empty vector clone L4440.


  4. Imaging controls

    1. Prepare plates with the hlh-1 RNAi clone B0304.1 (coordinates II-3J04 in the Ahringer library) and the empty vector clone L4440, as detailed above.

    2. Transfer L4 larvae from strain VBS662 or VBS663 to RNAi plates.

    3. Wait 24 to 48 h for uptake and processing of dsRNA through the RNAi pathway, then mount and image the progeny larvae. In worms exposed to hlh-1 dsRNA, you should observe strong and consistent nuclear localisation of fluorescently labelled NRDE-3 in all muscle cells. In a subset (~5%), you should observe one or two nuclear spots (Figure 4A). Nuclear localisation can be visible on a fluorescence dissecting scope. In worms exposed to the empty vector, you should observe constitutive nuclear localisation in the germline, early embryo, and intestine (see section “Nuclear localisation of NRDE-3”). All other tissues should show cytoplasmic localisation. In the early embryo, bright perinuclear spots are also visible.

    4. If observing the parental (P0) individuals that were transferred to the positive control plate, you should be able to observe nuclear localisation of NRDE-3 in muscle cells. However, double-stranded RNA will be unevenly distributed throughout the worm, and nuclear localisation will be weaker, and not observed in every muscle cell (Figure 4B).



      Figure 4. Labelling of hlh-1 active transcription sites.

      (A) Nuclear localisation of fluorescently labelled NRDE-3 in muscle cells and labelling of active hlh-1 transcription sites (arrowheads) in the progeny of individuals exposed to hlh-1 dsRNA. (B) Weak nuclear localisation of NRDE-3 in P0s exposed to hlh-1 dsRNA.


    Crossing in mutations

    It might be desirable to cross mutations or transgenes of interest in NRDE-3 strains. eri-1 (mg366) is located on chromosome IV; nrde-2(gg95) and YFP:NRDE-3 (at location ttTi5605) on chromosome II, 1.6 cM apart.

    1. Cross spontaneous males from VBS662, VBS663, VBS664, or VBS668 (eri-1(-) has a mild him phenotype) to hermaphrodites from the strain of interest.

    2. Isolate fluorescent F1s, which should be showing nuclear localisation of YFP or mNeonGreen::NRDE-3 in all tissues due to the eri-1(+).

    3. Let F1 hermaphrodites self-fertilise, and pick fluorescent F2.

    4. To homozygous in F2 and F3, pick hermaphrodites with cytoplasmic localisation of fluorescent signal in most tissues. It should be noted that the maternal effect of eri-1(+) can extend up to the F3 generation (Zhuang and Hunter, 2011).

    5. To homozygous nrde-2(gg95) and the fluorescent transgene, select lines that transmit 100%. Because nrde-2 and ttTi5605, the insertion site of fluorescent transgenes, are just 1.6 centiMorgans apart, it is easy to simply select 100% transmission of the fluorescent transgene. To select directly for nrde-2(-) homozygosity, select by resistance to lethal RNAi: RNAi targeting lir-1 (F18A1.3; clone II-5B14 from the Ahringer library), as in the original nrde screen (Guang et al., 2010), causes arrest in L1 in nrde-2(+). lir-1 is in an operon with lin-26; if nuclear RNAi is active, RNAi targeting lir-1 will also silence lin-26, causing L1 lethality (Bosher et al., 1999). Alternatively, primers CCTTCAAGTATCTATCCAGCTGCTCC/ GATCCAGTAGCCGAAGCTCTAGTTC can be used to track the gg95 mutation by PCR. The wild-type PCR product is 585 nucleotides long, while the mutant is 330 nucleotides long.


    Performing mutagenesis or transgenesis by CRISPR in those strains is possible. However, given that Cas9 is optimally active at 25°C, a temperature at which eri-1(-) induces sterility, it is best done in a wild-type background, before crossing it in.

    Extrachromosomal arrays, due to their repetitive nature, can cause simultaneous sense and antisense transcription. As a result, even in the absence of exposure to dsRNA, we can observe nuclear localisation and active transcription sites (Figure 5); they should be avoided if possible.



    Figure 5. Extrachromosomal arrays induce spurious labelling of transcription sites.

    Nuclear localisation and transcription site labelling (arrowheads) in individuals carrying a repetitive extrachromosomal array, without exposure to dsRNA.

    Data analysis

    No dedicated software package currently exists for this method. We use FIJI and the Trackmate software package (Tinevez et al., 2017) to segment active transcription sites and measure the fluorescence intensity of active transcription sites and the nuclear background.


    Nuclear localisation of NRDE-3

    Nuclear localisation of NRDE-3 signals that the gene of interest was expressed in this cell; however, it cannot tell how recently it was expressed. Once NRDE-3 has loaded a small RNA, it should not dissociate: the affinity of Argonautes to their guide RNA is rather high, with Kd on the order of 15 nM to 32 µM (MacRae et al., 2008; Wang et al., 2009). Once translocated to the nucleus, NRDE-3 stays nuclear even if no active transcription is ongoing. At this point, we would advise against using nuclear/cytoplasmic localisation to assess whether transcription is actively ongoing.

    We observe nuclear localisation in the gut, germline, and early embryo in the absence of double-stranded RNA exposure. We have nonetheless managed to observe active transcription sites in the gut after exposure to dsRNA targeting elt-2; in the germline and early embryo, we have so far failed.

    Other factors might affect nuclear localisation: low temperatures (15°C or 4°C) or UV irradiation can induce synthesis of risiRNAs, a class of triphosphorylated small RNAs loaded by NRDE-3 (Zhou et al., 2017), and cause nuclear localisation of NRDE-3 and formation of spots at nucleoli. It is possible that other undescribed stressors could induce risiRNA synthesis; therefore, unexpected nuclear localisation of NRDE-3 should be carefully interpreted.


    Limitations

    Several limitations of this method need to be highlighted. NRDE-3 based labelling does not work equally well in all tissues. The germline is so far refractory, and while labelling is possible in neurons, the efficiency is reduced. Labelling has not been successful for all genes (see Supplementary Table 1 in Toudji-Zouaz et al., 2021).

    Because the synthesis of secondary small RNAs is dependent on the presence of the target mRNA in the cytoplasm, this method is not able to label the first initiation of transcription. The length of this delay has not been estimated. While the nrde-2 mutation is sufficient to block transcriptional silencing, some downregulation by the primary RNAi pathway, necessary for synthesis of secondary small RNAs, is unavoidable. This can be mitigated by titrating down the concentration of dsRNA-expressing bacteria. Finally, while NRDE-3-bound transcripts are able to be exported to the cytoplasm (Figure 3), it is possible that they are affected in their processing, transport, or stability.

    Recipes

    1. 1 M KPO4 buffer

      108.3 g KH2PO4

      35.6 g K2HPO4

      ddH2O to 1 L

    2. M9 buffer

      3 g KH2PO4

      6 g Na2HPO4

      5 g NaCl

      1 mL of 1 M MgSO4

      ddH2O to 1 L

    Acknowledgments

    Funding: This work is funded by the Agence Nationale de la Recherche (ANR-11-LABX-0054, ANR-17-ERC2-0018 and ANR-21-CE13-0007) and the Fondation pour la Recherche Médicale (DEQ20180339160).

    This protocol was developed in a recent study (Toudji-Zouaz et al., 2021).

    Competing interests

    The authors declare no competing interests.

    References

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简介

[摘要]活性转录位点的实时标记对于我们理解转录动力学至关重要。在最广泛使用的方法中,将 RNA 序列 MS2 重复序列添加到感兴趣的转录本中,荧光标记的主要涂层蛋白在其上结合,并标记转录位点和转录本。在这里,我们描述了另一种策略,使用Argonaute蛋白 NRDE-3,重新用作 RNA可编程RNA结合蛋白。我们标记了秀丽隐杆线虫胚胎和幼虫中的活跃转录位点,而不编辑感兴趣的基因。 NRDE-3 是通过给线虫喂食与目标基因匹配的双链 RNA 来编程的。这种方法不需要基因组编辑,而且成本低廉且可快速应用于许多不同的基因。

图形概要:



[背景]我们在这里描述了一种 使用Argonaute蛋白 NRDE-3 在秀丽隐杆线虫转录位点成像转录的协议,基于我们最近发表的一种方法( Toudji-Zouaz 等人,2021) 。在秀丽隐杆线虫中,靶向感兴趣基因的双链 RNA被系统性 RNAi 机制吸收,并通过 dsRNA 转运蛋白 SID-1 分配给所有细胞。
exoRNAi途径将处理 dsRNA并将其用于识别目标 mRNA 以进行降解。这会将依赖于 RNA 的 RNA 聚合酶 RRF-1 募集到转录本中,这将合成三磷酸化的反义二级小 RNA,这些小 RNA 被加载到二级Argonautes中。唯一的体细胞次级Argonaute ,NRDE-3,当空载时,是细胞质的。一旦装载了小 RNA,NRDE-3 就会移动到细胞核并与新生转录物结合,在那里它会招募其他蛋白质来沉默转录。
在野生型背景下,内源性 RNAi 途径将诱导次级小 RNA 的合成和 NRDE-3 的组成型核定位(图 1A,B)。为了防止这种情况,我们在eri-1中引入了一个突变。因此,NRDE-3 在大多数组织(生殖系、肠道和早期胚胎除外)中是空载的,定位于细胞质,并且只有在感兴趣的基因在细胞中表达时才会进入细胞核( Guang 等。 , 2008) (图 1C, D)。为了防止下游转录沉默,我们还在nrde-2中引入了一个突变 (广 等。 , 2010) 。


图 1. NRDE-3 核定位依赖于小 RNA。
eri-1(+)遗传背景下,荧光标记的 NRDE-3 在胚胎 (A) 和成人体细胞组织 (头部) (B) 中的核定位。 NRDE-3 在eri-1(-)背景、胚胎 (C) 和成人 (D) 中的细胞质定位。注意胚胎 (C) 中种系原基的核定位。

关键字:转录标记, mRNA标记, 秀丽隐杆线虫, 船蛸属, 活体成像, 荧光显微镜

材料和试剂
1. 培养皿,直径 60 毫米(猎鹰, 目录号:353004)
2. 显微镜载玻片( Ghäasel ,目录号:29-201-307)
3. 盖玻片( Knittel ,目录号:100037)
4. 氯化钠(罗斯,CAS:7647-14-5)
5. 细菌琼脂 (BD, CAS: 9002-18-0)
6. Bacto蛋白胨 (BD, CAS: 51142-18-8)
7. 乙醇中的 5 mg/mL 胆固醇,过滤灭菌(Sigma,CAS:57-88-5)
8. KH 2 PO 4 (罗斯,CAS 7778-77-0)
9. K 2 HPO 4 (VWR,CAS:16788-57-1)
10. 1 M MgSO 4 (罗斯, CAS: 7487-88-9)
11. 1 M CaCl 2 (VWR, CAS: 10035-04-8)
12. 氨苄青霉素 1,000 × (Sigma, CAS: 69-53-4) 或羧苄青霉素 1,000 × (Roth, CAS: 4697-36-3) 25 mg/mL(-20°C 储存)
13. IPTG (Sigma, CAS: 367-93-1), 1 M (储存于-20°C)
14. Na 2 HPO 4 (VWR,CAS:7558-79-4)
15. 从文库( Ahringer文库,Source Bioscience;Vidal 文库,Horizon discovery,目录号: RCE1181;储存在 -80°C)或自制的靶向感兴趣基因的 RNAi 克隆喂养
16. 血清素(Sigma,目录号:H7752-5G),M9 中 25 mM 储备溶液,保持在-20°C
17. 0.1 µm 直径聚苯乙烯微球( Polysciences ,目录号:00876-15,M9 中 2.5% w/v 悬浮液)
18. 秀丽隐杆线虫的线虫菌株 遗传学 中心(明尼苏达大学):VBS662、VBS663、VBS664、VBS668
19. Wizard Plus SV 小量制备试剂盒(Promega,目录号:A1460)
20. LB 琼脂板含 50 µg/mL 氨苄青霉素和 10 µg/mL 四环素
21. 含 50 µg/mL 氨苄青霉素的液体 LB
22. 1 M KPO 4缓冲液(见配方)
23. M9 缓冲器(见配方)


设备


1. 蠕动泵(Wheaton Om nispense plus)
2. 共聚焦显微镜,Nikon Eclipse Ti 上的 Spinning disk Roper,带有用于 515 nm 照明的氩激光
3. 孵化器(Pol Eko 公寓),设置在 20°C
4. 离心机 (Eppendorf 5804R)
5. 蠕虫镐,根据( Stiernagle , 2006)准备


软件


1. 斐济 ( https://fiji.sc/ )


程序


此过程相对简单,几天即可实施(图 2)。它包括为表达 dsRNA 的细菌制备 RNAi 板、暴露线虫和成像。


 
图 2. 程序流程图。


准备板
用标准线虫生长培养基( Stiernagle , 2006)制备板 ,补充氨苄青霉素或羧苄青霉素(选择携带目的质粒的细菌)和 IPTG(诱导双链 RNA 转录)。通过喂食遵循 RNAi 的标准指南( Ahringer ,2006) 。在板制备过程中加入抗生素,而不是之后,增加了 dsRNA 表达的一致性。


A. NGM RNAi 板的制备
1. 在瓶子中混合 3 g NaCl、17 g 琼脂和 2.5 g 蛋白胨;添加 975 mL 的 ddH 2 O。高压灭菌 50 分钟。
2. 让瓶子冷却到 ~55°C。
3. 加入 1 mL 的 1 M CaCl 2 、1 mL 的 5 mg/mL 胆固醇、1 mL 的 1 M MgSO 4 、25 mL 的 1 M KPO 4缓冲液、1 mL 的 IPTG 和 1 mL 的羧苄青霉素或氨苄青霉素。
4. 在无菌条件下,用蠕动泵将盘子倒在火焰旁边(60 mm 直径盘子为 7 mL)。
5. 播种前,平板应在黑暗中至少晾干 24 小时;如果太湿,播种后细菌将无法正常干燥。为获得最佳实践,板应在两周内使用,尽管我们观察到用一个月大的板贴标签。


选择 RNAi 克隆
大肠杆菌菌株 HT115 细菌中的两个大型 RNAi 克隆文库可供使用: Ahringer文库(Kamath等人, 2003) ,Source Bioscience 不再商业销售,但可广泛使用,涵盖具有约 1 kb 长克隆的基因组序列;和 Vidal ORFeome库( Rual et al ., 2004) ,仍然由 Horizon Discovery 商业发行,包含完整的阅读框。如果您感兴趣的基因在这些文库中不可用,请通过在 L4440 空载体中克隆感兴趣的 ORF 来制作您自己的载体( Ahringer ,2006) 。
转录位点的标记依赖于荧光标记的 NRDE-3 在转录本上的平铺;它得出的结论是,喂给蠕虫的 dsRNA 越长,每个转录本上积累的荧光蛋白就越多。当我们测试仅针对hlh-1的一个外显子的 RNAi 克隆时,我们能够观察到 NRDE-3 在肌肉细胞中的核定位,但未能观察到活跃的转录位点。为了最大化信号,因此最好使用长 RNAi 克隆。 RdRP RRF -1 处理目标 RNA 3'→5';因此,目标转录本 3' 的 RNAi 克隆将诱导覆盖更多转录本的二级 siRNA。


B. RNAi 培养物的制备
1. 使用无菌移液器将冷冻文库中的条纹克隆到含有 50 µg/mL 氨苄青霉素(以选择是否存在 dsRNA 编码质粒)和 10 µg/mL 四环素(以选择 HT115 中dsRNAse中的突变)的 LB 板上尖端或接种环,并在 37°C 下生长过夜。
2. (可选)小量制备和测序克隆。这些库中的少数克隆被错误注释;因此,确认他们的身份是一种很好的做法。
3. 在 5 mL 的液体 LB 中培养 RNAi 克隆,加入 50 μg/mL 氨苄青霉素,在 37°C 和 200 rpm 下 8 小时至过夜。
4. 将培养物沉淀下来,倒出上清液,将沉淀重新悬浮在 200-300 µL中,并在板上播种 100 µL。 IPTG 应该在几个小时内诱导 dsRNA 的转录。我们通常在第二天或当盘子足够干燥时将蠕虫转移到盘子上。


尽管nrde-2的突变在转录水平上消除了二次沉默( Guang 等人, 2010;图吉-祖阿兹 等人,2021) ,对于一些 dsRNA 克隆(例如, ant-1.1 、 ama-1 ),我们观察到有害影响(不育、致死),可能是由于主要 RNAi 途径足以实现显着水平的敲低。通过用携带空载体 L4440 的细菌将 RNAi 培养物稀释 1/2 至 1/5,可以绕过这些有害影响。


选择使用哪种菌株
CGC 提供四种菌株;所有转基因通过CRISPR整合为单拷贝,位于基因座ttTi5605(染色体II)或ttTi4348(染色体I)。


VBS662 eri-1(mg366) IV; nrde-2(gg95) p eef-1A.1::YFP::nrde-3 II
在没有 RNAi 处理的情况下,YFP 信号在所有阶段的细胞质中都非常均匀。在强eef-1A.1启动子控制下的表达高;因此,细胞核中的背景信号会很强,而微弱的转录信号将难以观察到。 YFP 的光稳定性也较低,因此不太适合延时成像。


VBS663 eri-1(mg366) IV; nrde-2(gg95) p rps-27:: mNeonGreen ::flag::nrde-3 II
mNeonGreen倾向于在胚胎的细胞质中形成聚集体,因此在此阶段不太适合成像。在幼虫阶段,信号非常均匀。在较弱的er rps-27启动子控制下的表达水平确保了更高的信噪比。较高的光稳定性使其更适合长时间的延时拍摄。


VBS668 eri-1(mg366) IV; nrde-2(gg95) p eef-1A.1::YFP::nrde-3::SL2::sid-1 II
通过在所有组织中表达 dsRNA 转运蛋白sid-1 ,该菌株提高了神经元的效率(Calixto等, 2010) 。但是,它并没有达到其他体细胞组织的效率; RNAi 通路的几个组成部分似乎在神经元中稀疏表达(Cao等人, 2017) 。在以 GFP 为目标的 dsRNA 喂养的蠕虫中,我们观察到成虫神经元的核定位,但这在幼虫中效率较低。较低的效率意味着必须查看多个个体才能看到正确的核定位和转录标记。


VBS664 eri-1(mg366) IV; nrde-2(gg95) p eef-1A.1:: VenusC ::nrde-3 II; p eef-1A.1:: VenusN ::nrde-3 I Bipartite Venus:NRDE-3 允许在多个 NRDE-3 在同一转录本上积累时重建荧光;因此减少了细胞核和细胞质中的背景荧光。在那些表达感兴趣基因的细胞中,除了活跃的转录位点外,核质仍然可见:一旦金星被重组,它就不会再次解离;从转录本中卸载后,NRDE-3 将停留或转移回细胞核,从而产生一些背景核质信号。观察到一些自发重组与一些细胞中的细胞骨架有关。降低的背景允许观察细胞质转录物作为快速漂白的微弱斑点(图 3)。偶尔会观察到两个以上的核点;它们可能表明正在处理和出口的成绩单。


 
图 3. 用二分金星标记细胞质转录物。 
标记hlh-1转录位点(白色箭头)和细胞质转录物(红色箭头),在转录本上具有分裂的金星互补。


C. 暴露于 dsRNA 和成像
1. 将喂养良好的 L4 雌雄同体转移到 RNAi 板上。为了减少细菌污染,首先将蠕虫挑选到未播种的板上,然后将它们转移到 RNAi 板上。我们建议转移 ~20 雌雄同体。
2. 在 20°C 下将蠕虫留在板上至少 24 小时,最多 4 天。最佳暴露可能因基因和感兴趣的发育阶段而异;尝试几次曝光时间。
3. 对于胚胎,解剖妊娠雌雄同体并安装胚胎,在载玻片上的 2-5% 琼脂垫上的水中,注意尽可能限制细菌转移;添加盖玻片并密封边缘以防止蒸发(Walston 和 Hardin,2010) 。
4. 对于幼虫和成虫,安装在 5% 的琼脂垫上,在 M9 中加入 1 到 2 µL 聚苯乙烯珠和 2 µL 血清素进行固定(Lee等人,2019) ,注意避免细菌转移;添加盖玻片并密封边缘以防止蒸发。
5. 共聚焦显微镜上的图像,最好是旋转圆盘共聚焦显微镜,以减少光漂白。这些菌株中使用的荧光蛋白YFP、 mNeonGreen和 Venus 与 515 nm 的激发兼容。应调整激光强度和曝光以限制光漂白;我们尽量保持在 0.3 mW左右。我们使用 60 ×/1.20 NA 物镜进行图像采集,它提供足够的分辨率和足够长的工作距离,以及 0.5 µm的 Z 步长。
6. 您应该观察表达感兴趣基因的细胞中的核定位,并且在其中一些细胞中,有一个或两个点,对应于活跃的转录位点。如果使用具有多个旁系同源物的基因,或在多倍体组织(例如肠道或皮下组织)中工作,则可以观察到两个以上的点。转录大小斑点的直径应 <1 µm;尺寸应受衍射限制。理论上,一旦 NRDE-3 移动到细胞核,斑点应该变得可观察到。


在多代接触 dsRNA 后,很少观察到转录位点。因此,我们建议限制暴露于一两代人。
不鼓励使用其他固定方法:叠氮化钠是一种常见的代谢毒物,最好避免使用;虽然可以观察到转录点,但我们通常没有观察到叠氮化物的动态。左旋咪唑可以起作用,只要浓度足够低。
虽然落射荧光显微镜足以观察核定位,但不太可能成功地对转录点进行成像。共聚焦显微镜对于成功区分弱转录位点和背景是必要的。由于光毒性和缺氧,多个小时的成像对蠕虫造成了负担;使用mNeonGreen ::NRDE-3,数小时后,细胞质中会出现非特异性斑点。因此最好限制激光功率(<0.4 mW ) 并限制转移到载玻片上的细菌数量。


阳性和阴性对照
作为阳性对照,我们建议靶向基因hlh-1 ,编码MyoD直系同源物,在所有肌肉细胞中以中等水平表达。作为阴性对照,我们使用空载体克隆 L4440。


D. 成像控制
1. 使用hlh-1 RNAi 克隆 B0304.1( Ahringer库中的坐标 II-3J04)和空向量克隆 L4440 准备板,如上所述。
2. 将 L4 幼虫从菌株 VBS662 或 VBS663 转移到 RNAi 板。
3. 等待 24 至 48 小时,通过 RNAi 途径吸收和处理 dsRNA,然后安装并成像后代幼虫。在暴露于hlh-1 dsRNA 的蠕虫中,您应该观察到荧光标记的 NRDE-3 在所有肌肉细胞中的强烈且一致的核定位。在一个子集 (~5%) 中,您应该观察到一个或两个核点(图 4A)。在荧光解剖范围内可以看到核定位。在暴露于空载体的蠕虫中,您应该观察生殖系、早期胚胎和肠道中的组成型核定位(参见“NRDE-3 的核定位”部分)。所有其他组织应显示细胞质定位。在早期胚胎中,也可以看到明亮的核周斑点。
4. 如果观察转移到阳性对照板的亲本 (P0) 个体,您应该能够观察到 NRDE-3 在肌肉细胞中的核定位。然而,双链 RNA 将不均匀地分布在整个蠕虫中,并且核定位会较弱,并且不会在每个肌肉细胞中观察到(图 4B)。


 
图 4. hlh-1活性转录位点的标记。
(A) 肌肉细胞中荧光标记的 NRDE-3 的核定位和暴露于hlh-1 dsRNA的个体后代中活性hlh-1转录位点(箭头)的标记。 (B) NRDE-3 在暴露于hlh-1 dsRNA的 P0s 中的弱核定位。


突变交叉
在 NRDE-3 菌株中交叉感兴趣的突变或转基因可能是可取的。 eri-1 (mg366)位于第四染色体; nrde-2(gg95)和 YFP:NRDE-3 (在位置 ttTi5605) 在染色体 II 上,相距 1.6 cM 。
1. 从 VBS662、VBS663、VBS664 或 VBS668( eri-1(-)具有温和的 he 表型)到感兴趣菌株的雌雄同体的交叉自发雄性。
2. 由于eri-1(+) ,它应该在所有组织中显示 YFP 或mNeonGreen ::NRDE-3 的核定位。
3. 让 F1 雌雄同体自受精,并选择荧光 F2。
4. 为了在 F2 和 F3 中纯合,在大多数组织中选择具有细胞质定位荧光信号的雌雄同体。需要注意的是, eri-1(+)的母体效应可以延伸到 F3 代(Zhuang and Hunter, 2011) 。
5. 对于纯合nrde-2(gg95)和荧光转基因,选择传输 100% 的线。由于nrde-2和ttTi5605荧光转基因的插入位点相距仅1.6 centiMorgans ,因此很容易选择100%透光率的荧光转基因。要直接选择nrde-2(-)纯合性,请通过对致命 RNAi 的抗性进行选择: RNAi 靶向lir-1 (F18A1.3;来自Ahringer库的克隆 II-5B14),如原始nrde筛选( Guang et al ., 2010) ,导致nrde-2(+)中的 L1 停滞。 lir-1在一个带有lin-26的操作子中;如果核 RNAi 是活跃的,靶向lir-1 的RNAi 也会使lin-26沉默,导致 L1 致死(Bosher等人, 1999) 。或者,引物 CCTTCAAGTATCTATCCAGCTGCTCC/GATCCAAGTAGCCGAAGCTCTAGTTC 可用于通过 PCR 跟踪gg95突变。野生型 PCR 产物长 585 个核苷酸,而突变型 PCR 产物长 330 个核苷酸。


通过 CRISPR 在这些菌株中进行诱变或转基因是可能的。然而,鉴于 Cas9 在 25°C( eri-1(-)诱导不育的温度)下具有最佳活性,因此最好在野生型背景下进行,然后再将其插入。
染色体外阵列,由于其重复性,可以同时引起有义和反义转录。因此,即使在没有接触 dsRNA 的情况下,我们也可以观察到核定位和活跃的转录位点(图 5);如果可能,应避免使用它们。


 
图 5. 染色体外阵列诱导转录位点的虚假标记。 
携带重复染色体外阵列的个体中的核定位和转录位点标记(箭头),未暴露于 dsRNA。


数据分析_


当前不存在此方法的专用软件包。我们使用 FIJI 和Trackmate软件包( Tinevez et al ., 2017)分割活性转录位点并测量活性转录位点的荧光强度和核背景。


NRDE-3的核定位
NRDE-3 的核定位表明感兴趣的基因在该细胞中表达;但是,它无法说明它是如何最近表达的。一旦 NRDE-3 加载了一个小 RNA,它就不会解离: Argonautes与其向导 RNA 的亲和力相当高, K d约为 15 nM至 32 µM ( MacRae 等人, 2008;王等人, 2009) 。一旦转移到细胞核中,即使没有进行活跃的转录,NRDE-3 也会保持在细胞核中。在这一点上,我们建议不要使用核/细胞质定位来评估转录是否正在积极进行。
在没有双链 RNA 暴露的情况下观察到肠道、种系和早期胚胎中的核定位。尽管如此,我们还是设法在暴露于靶向elt-2的 dsRNA 后观察到肠道中的活跃转录位点;在生殖系和早期胚胎中,我们迄今为止都失败了。
其他因素可能会影响核定位:低温(15°C 或 4°C)或紫外线照射可诱导 risiRNAs 的合成, risiRNAs是由 NRDE-3 加载的一类三磷酸化小 RNA (Zhou et al ., 2017) ,并导致NRDE-3的核定位和核仁斑点的形成。其他未描述的压力源可能会诱导risiRNA合成。因此,应仔细解释 NRDE-3 的意外核定位。


限制
需要强调这种方法的几个局限性。基于 NRDE-3 的标记并非在所有组织中都同样有效。迄今为止,种系是难治的,虽然可以在神经元中进行标记,但效率会降低。并非所有基因都成功标记(参见Toudji-Zouaz中的补充表 1 等人,2021 年)。
因为次级小 RNA 的合成依赖于细胞质中靶 mRNA 的存在,所以这种方法不能标记转录的第一个起始。这种延迟的长度尚未估计。虽然nrde-2突变足以阻止转录沉默,但初级 RNAi 途径的一些下调是不可避免的,这是合成次级小 RNA 所必需的。这可以通过降低表达 dsRNA 的细菌的浓度来缓解。最后,虽然 NRDE-3 结合的转录本能够被输出到细胞质中(图 3),但它们的加工、运输或稳定性可能会受到影响。


食谱


1. 1 M KPO 4缓冲液
108.3 克 KH 2 PO 4
35.6 克 K 2 HPO 4
ddH 2 O 至 1 L
2. M9缓冲器
3克KH 2 PO 4
6 克 Na 2 HPO 4
5克氯化钠
1 mL 1 M MgSO 4
ddH 2 O 至 1 L


致谢


资助:这项工作由机构资助 Nationale de la Recherche (ANR-11-LABX-0054, ANR-17-ERC2-0018 and ANR-21-CE13-0007) 和Fondation pour la Recherche Médicale (DEQ20180339160)。
该协议是在最近的一项研究中开发的( Toudji-Zouaz 等人,2021)。


利益争夺


作者声明没有竞争利益。


参考


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引用:Barrière, A. and Bertrand, V. (2022). Labelling of Active Transcription Sites with Argonaute NRDE-3—Image Active Transcription Sites in vivo in Caenorhabditis elegans. Bio-protocol 12(11): e4427. DOI: 10.21769/BioProtoc.4427.
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