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RNase Sensitivity Screening for Nuclear Bodies with RNA Scaffolds in Mammalian Cells
哺乳动物细胞中含RNA支架的核体的核糖核酸酶敏感性筛选   

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参见作者原研究论文

本实验方案简略版
The Journal of Cell Biology
Jul 2016

Abstract

The mammalian cell nucleus is highly organized and contains membraneless nuclear bodies (NBs) characterized by distinct resident factors. The NBs are thought to serve as sites for biogenesis and storage of certain RNA and protein factors as well as assembly of ribonucleoprotein complexes. Some NBs are formed with architectural RNAs (arcRNAs) as their structural scaffolds and additional NBs likely remain unidentified in mammalian cells. Here, we describe an experimental protocol to search for new NBs built on certain arcRNAs. RNase-sensitive NBs were identified by monitoring nuclear foci visualized by tagging thousands of human cDNA products.

Keywords: Nuclear bodies (核体), Architectural RNA (结构RNA), RNase (核糖核酸酶), Human full-length cDNA library (人全长cDNA文库), Venus tag (Venus标签), Subcellular localization (亚细胞定位)

Background

The mammalian cell nucleus is highly organized and composed of multiple distinct substructures called nuclear bodies (NBs). So far ~15 NBs have been identified as subnuclear membraneless granular structures containing various proteins and RNA factors, many of which function as sites of biogenesis, maturation, storage, and sequestration of specific RNAs, proteins, and/or ribonucleoprotein (RNP) complexes (Mao et al., 2011; Sleeman and Trinkle-Mulcahy, 2014) (Table 1).

Table 1. Nuclear bodies in mammalian cells


Some NBs are constructed on specific long noncoding RNAs (lncRNAs) called architectural RNAs (arcRNAs), which are defined as structural core of NBs (Chujo et al., 2016). The arcRNA-dependent NBs are composed of numerous RNA-binding proteins that interact with the arcRNAs. The most remarkable example is the paraspeckle, which is composed of several characteristic RNA-binding proteins (Fox et al., 2002; Prasanth et al., 2005). RNase treatment disintegrates the paraspeckle structure (Fox et al., 2005). Nuclear paraspeckle assembly transcript 1 (NEAT1), a lncRNA, localizes exclusively to paraspeckles and acts as an arcRNA of these massive RNP complexes (Chen and Carmichael, 2009; Clemson et al., 2009; Sasaki et al., 2009; Sunwoo et al., 2009).

Presently, two lncRNAs are classified as arcRNAs in addition to NEAT1, namely, intergenic spacer lncRNAs for nucleolar detention center (Audas et al., 2012) and human satellite III lncRNA for nuclear stress body (Biamonti and Vourc’h, 2010) (Table 1). It is expected that additional arcRNAs remain to be characterized in mammalian cells. Here, we describe a novel method called ‘RNase sensitivity screening’ to identify novel arcRNA-dependent NBs by screening for nuclear foci whose structures are disintegrated by RNase treatment. This method employed a Venus-tagged human full-length (FLJ) cDNA library (32,651 clones), which was originally constructed during the NEDO full-length human cDNA sequencing project in Japan (FLJ-PJ), and obtained 571 cDNA clones whose products (463 proteins) localize in certain nuclear foci (Hirose and Goshima, 2015; Naganuma et al., 2012). We explored whether the respective nuclear focus was abolished or diffused upon RNase treatment after cell permeabilization to select candidate RNase-sensitive nuclear foci that potentially contain arcRNAs (Figure 1). We identified 32 Venus-tagged proteins that required RNA for their localization in distinct nuclear foci (Mannen et al., 2016). Immunostaining of the corresponding endogenous proteins confirmed that the Sam68 nuclear body (SNB) was an RNase-sensitive NB. In the following protocol, we describe the detailed procedure of the RNase sensitivity screening. This protocol is for screening for RNase-sensitive NBs under normal conditions in HeLa cells, but it should be applicable to other cell lines under various conditions.


Figure 1. Outline of RNase sensitivity screening of NBs. Venus-tagged human FLJ cDNA clones were transfected into HeLa cells. cDNA clones whose products localized to certain nuclear foci were selected (571 clones). Subsequently, the RNase sensitivity of the nuclear foci labeled by Venus was investigated (32 clones). To this end, the cells were permeabilized with 2% Tween 20, followed by treatment with an RNase mixture. Bar = 10 μm.

Materials and Reagents

Note: Prepare all solutions using ultrapure water (prepared by purifying deionized water to attain a sensitivity of 18.2 MΩ cm at 25 °C) and analytical grade reagents.

  1. Preparation of collagen IV-coated plate
    1. Pipette tips (Labcon, catalog number: 1030-260-000 )
    2. 96-well glass bottom microplate (IWAKI, catalog number: 12-017-006 )
    3. Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 190-14565 )
    4. Phosphate Buffered Salts (PBS) (Takara Bio, catalog number: T900 )
    5. Hydrochloric acid (HCl) (Wako Pure Chemical Industries, catalog number: 080-01066 )
    6. Type IV collagen solution (Life Laboratory Company, catalog number: LL-10043 )
    7. Collagen solution (see Note 1; see Recipes)

  2. Cell culture
    1. HeLa (Human cervical cancer) cells (see Note 2)
    2. 10 cm dish
    3. MEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11095080 ) (see Note 3)
    4. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
    5. Trypsin-EDTA (Nacalai Tesque, catalog number: 32778-34 )

  3. Administration of plasmids with transfection reagent
    1. 96-well U-bottom plate (Corning, Falcon®, catalog number: 351177 )
    2. Venus-tagged human FLJ cDNA clone plasmids expressing proteins localized to certain nuclear foci (see Note 4)
    3. TransIT-LT1 reagent (Mirus Bio, catalog number: MIR2300 )
    4. Opti-MEM I reduced serum medium (Thermo Fisher Scientific, GibcoTM, catalog number: 31985070 )

  4. RNase treatment
    1. 96-well plate sealing film (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12261012 )
    2. 0.45 μm filter
    3. RiboShredder RNase Blend (Epicentre, catalog number: RS12500 )
    4. Polyoxyethylene sorbitan monolaurate (Tween 20) (Nacalai Tesque, catalog number: 28353-85 )
    5. 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris) (Wako Pure Chemical Industries, catalog number: 011-20095 )
    6. Magnesium chloride (MgCl2) (Nacalai Tesque, catalog number: 20909-55 )
    7. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Nacalai Tesque, catalog number: 08907-42 )
    8. cOmplete, EDTA-free protease inhibitor cocktail (Roche Diagnostics, catalog number: 11873580001 )
    9. Paraformaldehyde (PFA) (EMD Millipore, catalog number: 1.04005.1000 )
    10. DAPI (Sigma-Aldrich, catalog number: D9542 )
    11. Pyronin Y (Sigma-Aldrich, catalog number: P9172 )
    12. 1 M Tris-HCl, pH 7.4 (see Recipes)
    13. Permeabilization buffer (see Recipes)
    14. 4% PFA (see Note 5; see Recipes)
    15. DAPI solution (see Recipes)
    16. Pyronin Y solution (see Recipes)

Equipment

  1. Multichannel pipette L8 x 200 (Gilson, catalog number: FA10011 )
  2. Water bath (Fine, catalog number: FWB-21B )
  3. Centrifuge (KUBOTA, catalog number: 040-000 )
  4. Hemocytometer (SANSYO, SLGC, catalog number: A103 )
  5. Reservoir (BM Equipment, catalog number: BM-0850-2 )
  6. Carbon dioxide (CO2) incubator (SANYO, catalog number: MCO-18AIC UV )
  7. IN Cell Analyzer 1000 (GE Healthcare, model: IN Cell Analyzer 1000 ) equipped with a Plan Fluor ELWD 40x/0.6 objective lens (Nikon Instruments, model: CFI Plan Fluor 40x ). Excitation filters for DAPI (D360/40x) and Venus (S475/20x) and emission filters for DAPI (HQ460/40M) and Venus (HD535/50M) were used. Acquisition and processing of the images were done using IN Cell Analyzer 1000 Software (GE Healthcare, version 3.0)

Software

  1. IN Cell Analyzer 1000 Software (GE Healthcare, version 3.0)

Procedure

  1. Preparation of collagen-coated plate
    1. Wash each well of 96-well glass bottom microplates with 100 μl 0.2 N NaOH at room temperature for 30 min.
    2. Rinse each well with 100 μl PBS and then 100 μl distilled water.
    3. Coat the glass bottom of each well with 100 μl collagen solution at room temperature for 1 h (see Note 6).
    4. Wash each well with 100 μl PBS twice.
    5. Remove PBS completely from each well by pipetting. The plates can be stored at 4 °C for 1 week.

  2. Cell culture
    1. Thaw frozen cell stocks in a water bath at 37 °C, and retrieve the cells by low speed centrifugation (220 x g, 5 min). Remove the supernatant by pipetting.
    2. Gently resuspend the cell pellet in 10 ml growth medium (e.g., MEM medium supplemented with 10% FBS) without antibiotics.
    3. Culture the cells in growth medium in a humidified incubator with 5% CO2 until 80% confluency is reached.
    4. For plasmid transfection, seed the cells into a 10 cm dish 1 day prior to use (see Note 7).

  3. Administration of plasmids with transfection reagent
    1. Seed 6 x 103 cells into 100 μl growth medium in each well of a collagen-coated 96-well plate 1 day prior to transfection. The cells should be used for transfection when they have reached 30-50% confluency.
    2. Add 100 ng Venus-tagged FLJ cDNA clone plasmids to 50 μl Opti-MEM I reduced medium in each well of a 96-well U-bottom plate.
    3. Add 0.3 μl TransIT-LT1 reagent to 50 μl Opti-MEM I reduced medium in each well of a separate 96-well U-bottom plate.
    4. Mix the solutions prepared in steps C2 and C3 and incubate at room temperature for 15-30 min to form the plasmid-TransIT-LT1 complexes.
    5. Add the resultant plasmid-TransIT-LT1 complexes (ca. 100 μl) directly to the growth medium in each well of the 96-well plate prepared in step C1.
    6. Culture the cells for 24 h in the incubator with 5% CO2 (see Note 8).

  4. RNase treatment
    1. Remove the culture medium by pipetting and wash the cells once with 100 μl PBS.
    2. Wash the cells once with 100 μl permeabilization buffer and then permeabilize the cells with 100 μl 2% Tween 20-permeabilization buffer at room temperature for 10 min (see Note 9).
    3. Wash the cells once with 100 μl permeabilization buffer.
    4. Wash the cells once with 100 μl PBS and degrade RNAs with 100 μl 50 U/ml RiboShredder RNase Blend-PBS at room temperature for 20 min (see Note 10).
    5. Wash the cells twice with 100 μl PBS.
    6. Fix the cells with 100 μl 4% PFA at room temperature for 15 min.
    7. Wash the cells three times with 100 μl PBS for 5 min each.
    8. Stain the DNA with 100 μl DAPI solution for 1 min.
    9. Wash the cells three times with 100 μl PBS.
    10. (Optional, see Note 11) Stain the RNA with 100 μl Pyronin Y solution for 15 sec.
    11. (Optional) Wash the cells three times with 100 μl PBS.
    12. Add 100 μl PBS to each well and seal the wells with a 96-well plate sealing film.
    13. Visualize Venus signals and detect nuclear foci in the samples using the IN Cell Analyzer 1000 (Figure 2). The images were acquired under 400x magnification, with automatically optimized exposure times and hardware autofocus. Images of 25 different fields were acquired from each well. (see Note 12)


      Figure 2. Images of RNase sensitivity screening of NBs. Effect of RNase treatment was confirmed by visualizing cellular RNAs with Pyronin Y staining, and by monitoring the persistence of the RNase-resistant Cajal bodies (marked by COIL-Venus) and disappearance of the RNase-sensitive paraspeckles (marked by SFPQ-Venus). Sam68 nuclear body is a newly identified RNase-sensitive NB. Arrowheads indicate nuclear foci. DNA was stained with DAPI (blue). Bar = 10 μm.

Data analysis

Cell numbers (n > 100) were determined by counting the outlined DAPI positive oval area as the nucleus using IN Cell Analyzer 1000 Software. The number of nuclear foci-positive cells was manually counted. RNase sensitivity is measured by the ratio of the number of nuclear foci-positive cells in RNase-treated cells relative to the number of those in the cells without RNase treatment.

Notes

  1. This solution should be prepared at the time of use.
  2. Although the protocol described here is for HeLa cells, it may be applicable to other cultured cell lines (e.g., HCT116, U2OS, and NIH3T3). These cell lines are available from several cell stock centers, such as the American Type Culture Collection.
  3. Culture medium should be properly chosen for the cell lines used.
  4. The list of FLJ clones expressing the Venus-tagged proteins that localized to certain nuclear foci is available at the following website: http://hgpd.lifesciencedb.jp/sys_info/download.html. The localization images of the Venus-tagged human cDNA products are accessible at the Human Gene and Protein Database (HGPD). The original Gateway Entry clones are available from the Biological Resource Center, National Institute of Technology and Evaluation (NBRC). We used two Venus-tagged proteins as controls: SFPQ-Venus (a marker of the paraspeckle, an RNase-sensitive NB) and COIL-Venus (a marker of the Cajal body, an RNase-resistant NB).
  5. This solution should be prepared at the time of use.
  6. Other coating reagents such as cationic polymers (e.g., poly-L-lysine) or matrix proteins (e.g., laminin, fibronectin) can be selected according to the cells used.
  7. The number of cell passages should be less than five.
  8. Transfection conditions should be optimized by varying the concentration of TransIT-LT1 reagent.
  9. Permeabilization conditions should be optimized by monitoring the control Venus-tagged proteins (Note 4) and Pyronin Y staining. The concentration of Tween 20 should be optimized for each cell line used. It should be noted that treatment with higher concentrations of Tween 20 often results in disappearance of Venus signals in the cells.
  10. RNase conditions should be optimized by monitoring the control Venus-tagged proteins (Note 4) and Pyronin Y staining. We always compare two samples with and without RNase treatment for one cDNA clone.
  11. Effects of RNase treatment are confirmed by staining of cellular RNAs with Pyronin Y.
  12. For final judgment of localization sites of the selected cDNA products, both N-terminally and C-terminally Venus-tagged proteins should be confirmed to consistently localize in identical NBs. Furthermore, the localization of the corresponding endogenous proteins should be confirmed by labeling with specific antibodies.

Recipes

  1. Collagen solution
    Dilute 8.6 μl/ml collagen in 0.05 N HCl
  2. 1 M Tris-HCl, pH 7.4 (1 L)
    Dilute 121.1 g Tris in ultrapure water and adjust pH to 7.4 with HCl
  3. Permeabilization buffer
    20 mM Tris-HCl, pH 7.4
    5 mM MgCl2
    0.5 mM EGTA
    1x cOmplete, EDTA-free protease inhibitor cocktail
  4. 4% PFA
    Add 0.4 g PFA to PBS and adjust the volume to 10 ml
    Shake vigorously at 55 °C until completely dissolved and filtrate through a 0.45 μm filter
  5. DAPI solution
    Add 10 mg DAPI to ultrapure water and adjust the volume to 1 ml (10 mg/ml DAPI stock)
    Store at -20 °C
    Before using, dilute 1 μl 10 mg/ml DAPI stock in 10 ml PBS (1 μg/ml DAPI)
  6. Pyronin Y solution
    Add 3 mg Pyronin Y to ultrapure water and adjust the volume to 10 ml (1 mM Pyronin Y stock)
    Store at -20 °C
    Before using, dilute 1,000 times with PBS (1 μM Pyronin Y)

Acknowledgments

This protocol was adapted from our recently published work (Mannen et al., 2016). We thank N. Goshima, Y. Kawamura, and H. Mochizuki at the National Institute of Advanced Industrial Science and Technology for their support in the preparation of the Venus-tagged cDNA library. This research was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (26113002 and 26291001).

References

  1. Audas, T. E., Jacob, M. D. and Lee, S. (2012). Immobilization of proteins in the nucleolus by ribosomal intergenic spacer noncoding RNA. Mol Cell 45(2): 147-157.
  2. Biamonti, G. and Vourc'h, C. (2010). Nuclear stress bodies. Cold Spring Harb Perspect Biol 2(6): a000695.
  3. Chen, L. L. and Carmichael, G. G. (2009). Altered nuclear retention of mRNAs containing inverted repeats in human embryonic stem cells: functional role of a nuclear noncoding RNA. Mol Cell 35(4): 467-478.
  4. Chujo, T., Yamazaki, T. and Hirose, T. (2016). Architectural RNAs (arcRNAs): A class of long noncoding RNAs that function as the scaffold of nuclear bodies. Biochim Biophys Acta 1859(1): 139-146.
  5. Clemson, C. M., Hutchinson, J. N., Sara, S. A., Ensminger, A. W., Fox, A. H., Chess, A. and Lawrence, J. B. (2009). An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol Cell 33(6): 717-726.
  6. Fox, A. H., Bond, C. S. and Lamond, A. I. (2005). P54nrb forms a heterodimer with PSP1 that localizes to paraspeckles in an RNA-dependent manner. Mol Biol Cell 16(11): 5304-5315.
  7. Fox, A. H., Lam, Y. W., Leung, A. K., Lyon, C. E., Andersen, J., Mann, M. and Lamond, A. I. (2002). Paraspeckles: a novel nuclear domain. Curr Biol 12(1): 13-25.
  8. Hirose, T. and Goshima, N. (2015). Genome-wide co-localization screening of nuclear body components using a fluorescently tagged FLJ cDNA clone library. Methods Mol Biol 1262: 155-163.
  9. Mannen, T., Yamashita, S., Tomita, K., Goshima, N. and Hirose, T. (2016). The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL. J Cell Biol 214(1): 45-59.
  10. Mao, Y. S., Zhang, B. and Spector, D. L. (2011). Biogenesis and function of nuclear bodies. Trends Genet 27(8): 295-306.
  11. Naganuma, T., Nakagawa, S., Tanigawa, A., Sasaki, Y. F., Goshima, N. and Hirose, T. (2012). Alternative 3’end processing of long noncoding RNA initiates construction of nuclear paraspeckles. EMBO J 31(20): 4020-4034.
  12. Prasanth, K. V., Prasanth, S. G., Xuan, Z., Hearn, S., Freier, S. M., Bennett, C. F., Zhang, M. Q. and Spector, D. L. (2005). Regulating gene expression through RNA nuclear retention. Cell 123(2): 249-263.
  13. Sasaki, Y. T., Ideue, T., Sano, M., Mituyama, T. and Hirose, T. (2009). MENepsilon/beta noncoding RNAs are essential for structural integrity of nuclear paraspeckles. Proc Natl Acad Sci U S A 106(8): 2525-2530.
  14. Sleeman, J. E. and Trinkle-Mulcahy, L. (2014). Nuclear bodies: new insights into assembly/dynamics and disease relevance. Curr Opin Cell Biol 28: 76-83.
  15. Sunwoo, H., Dinger, M. E., Wilusz, J. E., Amaral, P. P., Mattick, J. S. and Spector, D. L. (2009). MEN epsilon/beta nuclear-retained non-coding RNAs are up-regulated upon muscle differentiation and are essential components of paraspeckles. Genome Res 19(3): 347-359.

简介

哺乳动物细胞核高度组织,包含以不同的居民因素为特征的无膜核体(NBs)。 NB被认为是用于某些RNA和蛋白质因子的生物发生和储存的位点以及核糖核蛋白复合物的组装。 一些NB由构建的RNA(arcRNA)形成,作为它们的结构性支架,另外的NB可能在哺乳动物细胞中保持不明。 在这里,我们描述了一个实验协议来搜索建立在某些arcRNA上的新NB。 通过监测通过标记数千个人类cDNA产物可视化的核病灶来鉴定RNase敏感性NB。
【背景】哺乳动物细胞核是高度组织的,由称为核体(NB)的多个不同的亚结构组成。迄今为止,已经将15个NB鉴定为含有各种蛋白质和RNA因子的亚核膜无颗粒结构,其中许多颗粒结构用作特异性RNA,蛋白质和/或核糖核蛋白(RNP)复合物的生物发生,成熟,储存和螯合的位点Mao et al。,2011; Sleeman and Trinkle-Mulcahy,2014)(表1)。
一些NB被构建在称为结构RNA(arcRNA)的特定长非编码RNA(lncRNA)上,其定义为NB的结构核心(Chujo等,2016)。 arcRNA依赖的NB由与arcRNA相互作用的许多RNA结合蛋白组成。最显着的例子是由几种特征性RNA结合蛋白组成的寄生虫斑(Fox等,2002; Prasanth等,2005)。核糖核酸酶处理分解了斑点结构(Fox等,2005)。核霹雳组合转录物1(NEAT1)是一种lncRNA,仅专门用于发生突触并作为这些大规模RNP复合物的弧形RNA(Chen和Carmichael,2009; Clemson等人,2009; Sasaki等人,2009; Sunwoo等人,,2009)。
目前,除了NEAT1之外,还有两种lncRNA被归类为arcRNA,即用于核仁拘留中心的基因间隔核苷酸(Audas等,2012)和核应激体的人类卫星IIInnRNA(Biamonti和Vourc'h,2010)(表1)。预期在哺乳动物细胞中仍然表征额外的弧形RNA。在这里,我们描述了一种称为“RNase敏感性筛选”的新方法,通过筛选其核结构被核糖核酸酶处理分解的核病灶来鉴定新的arcRNA依赖性NB。该方法采用最初在日本NEDO全长人cDNA测序项目(FLJ-PJ)构建的维纳斯标记的人全长cDNA文库(32,651个克隆),获得了571个cDNA克隆,其产物(463个蛋白质)在某些核病灶中定位(Hirose和Goshima,2015; Naganuma等,2012)。我们探讨了在细胞透化后核糖核酸酶处理是否废除或分散各自的核重点,以选择潜在含有弧线氨基酸的候选核糖核酸酶敏感核病灶(图1)。我们确定了32种维纳斯标记蛋白,需要RNA在不同的核病灶中进行定位(Mannen等,2016)。相应的内源蛋白的免疫染色证实了Sam68核体(SNB)是RNase敏感型NB。在以下协议中,我们将描述RNase敏感性筛选的详细步骤。该方案用于在HeLa细胞中正常条件下筛选RNase敏感型NB,但应适用于各种条件下的其他细胞系。
图1. NBs的RNase敏感性筛选概述。将金星标记的人FLJ cDNA克隆转染入HeLa细胞。选择产品定位于某些核病灶的cDNA克隆(571个克隆)。随后,研究了由金星标记的核病灶的核糖核酸酶敏感性(32个克隆)。为此,将细胞用2%吐温20透化,然后用RNase混合物处理。 Bar =10μm。

关键字:核体, 结构RNA, 核糖核酸酶, 人全长cDNA文库, Venus标签, 亚细胞定位

材料和试剂

注意:使用超纯水(通过净化去离子水制备,在25℃下达到18.2MΩcm的灵敏度)和分析级试剂来制备所有溶液。

  1. 胶原IV涂层板的制备
    1. 移液器提示(Labcon,目录号:1030-260-000)
    2. 96孔玻璃底板(IWAKI,目录号:12-017-006)
    3. 氢氧化钠(NaOH)(Wako Pure Chemical Industries,目录号:190-14565)
    4. 磷酸缓冲盐(PBS)(Takara Bio,目录号:T900)
    5. 盐酸(HCl)(Wako Pure Chemical Industries,目录号:080-01066)
    6. IV型胶原溶液(Life Laboratory Company,目录号:LL-10043)
    7. 胶原蛋白溶液(见注1;参见食谱)

  2. 细胞培养
    1. HeLa(人类宫颈癌)细胞(见注2)
    2. 10厘米盘
    3. MEM培养基(Thermo Fisher Scientific,Gibco TM,目录号:11095080)(参见注3)
    4. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:26140079)
    5. 胰蛋白酶-EDTA(Nacalai Tesque,目录号:32778-34)

  3. 用转染试剂管理质粒
    1. 96孔U型底板(Corning,Falcon ®,目录号:351177)
    2. 金星标记的人类FLJ cDNA克隆质粒,其表达定位于某些核病灶的蛋白质(见附注4)
    3. IT-LT1试剂(Mirus Bio,目录号:MIR2300)
    4. Opti-MEM I减少血清培养基(Thermo Fisher Scientific,Gibco TM,目录号:31985070)

  4. 核糖核酸酶处理
    1. 96孔板密封膜(Thermo Fisher Scientific,Invitrogen TM,目录号:12261012)
    2. 0.45μm过滤器
    3. RiboShredder RNase Blend(Epicentre,目录号:RS12500)
    4. 聚氧乙烯脱水山梨醇单月桂酸酯(吐温20)(Nacalai Tesque,目录号:28353-85)
    5. 2-氨基-2-羟甲基-1,3-丙二醇(Tris)(Wako Pure Chemical Industries,目录号:011-20095)
    6. 氯化镁(MgCl 2)(Nacalai Tesque,目录号:20909-55)
    7. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Nacalai Tesque,目录号:08907-42)
    8. 完全,不含EDTA的蛋白酶抑制剂混合物(Roche Diagnostics,目录号:11873580001)
    9. 多聚甲醛(PFA)(EMD Millipore,目录号:1.04005.1000)
    10. DAPI(Sigma-Aldrich,目录号:D9542)
    11. Pyronin Y(Sigma-Aldrich,目录号:P9172)
    12. 1M Tris-HCl,pH 7.4(参见食谱)
    13. 渗透缓冲液(见配方)
    14. 4%PFA(见附注5;见配方)
    15. DAPI解决方案(见配方)
    16. Pyronin Y溶液(参见食谱)

设备

  1. 多通道移液器L8 x 200(Gilson,目录号:FA10011)
  2. 水浴(精细,目录号:FWB-21B)
  3. 离心机(KUBOTA,目录号:040-000)
  4. 血细胞计数器(SANSYO,SLGC,目录号:A103)
  5. 水库(BM设备,目录号:BM-0850-2)
  6. 二氧化碳(CO 2)培养箱(SANYO,目录号:MCO-18AIC UV)
  7. 配备有Plan Fluor ELWD 40x/0.6物镜(Nikon Instruments,型号:CFI Plan Fluor 40x)的IN Cell Analyzer 1000(GE Healthcare,型号:IN Cell Analyzer 1000)。使用DAPI(D360/40x)和金星(S475/20x)和DAPI(HQ460/40M)和金星(HD535/50M)的发射滤光片的激发滤光片。使用IN Cell Analyzer 1000软件(GE Healthcare,3.0版)
    完成图像的采集和处理

软件

  1. IN Cell Analyzer 1000软件(GE Healthcare,3.0版)

程序

  1. 胶原蛋白涂层板的制备
    1. 用100μl0.2N NaOH在室温下清洗96孔玻璃底部微孔板的每个孔30分钟
    2. 用100μlPBS冲洗每个孔,然后用100μl蒸馏水冲洗
    3. 每个孔的玻璃底部用100μl胶原溶液在室温下涂覆1小时(见附注6)
    4. 每孔用100μlPBS洗涤两次。
    5. 通过移液从每个孔中完全清除PBS。板可以在4℃下储存1周。

  2. 细胞培养
    1. 在37℃的水浴中解冻冷冻的细胞库,并通过低速离心(220×g,5分钟)回收细胞。通过移液去除上清液。
    2. 轻轻重悬细胞沉淀在10ml生长培养基(例如,补充有10%FBS的MEM培养基)中,不含抗生素。
    3. 在具有5%CO 2的湿润培养箱中培养生长培养基中的细胞,直至达到80%融合。
    4. 对于质粒转染,在使用前1天将细胞种植在10厘米的培养皿中(见注释7)。

  3. 用转染试剂管理质粒
    1. 种子6×10 3个细胞在转染前1天在胶原包被的96孔板的每个孔中的100μl生长培养基中。当细胞达到30-50%融合时,细胞应用于转染。
    2. 将100ng金星标记的FLJ cDNA克隆质粒加入到96孔U底板的每个孔中的50μlOpti-MEM I还原培养基中。
    3. 在单独的96孔U型底板的每个孔中加入0.3μlTrans IT-LT1试剂至50μlOpti-MEM I还原培养基。
    4. 混合步骤C2和C3中制备的溶液,并在室温下孵育15-30分钟以形成质粒-TransIT-LT1复合物。
    5. 将所得质粒-TransIT-LT1复合物(约100μl)直接加入到步骤C1中制备的96孔板的每个孔中的生长培养基中。
    6. 在5%CO 2培养箱中培养细胞24小时(见附注8)。

  4. 核糖核酸酶处理
    1. 用100μlPBS移液并清洗细胞一次,取出培养基
    2. 用100μl透化缓冲液洗涤细胞一次,然后用100μl2%吐温20-透化缓冲液在室温下透化细胞10分钟(见注9)。
    3. 用100μl透化缓冲液清洗细胞一次。
    4. 用100μlPBS洗涤细胞一次,并在室温下用100μl50U/ml RiboShredder RNase Blend-PBS将RNA降解20分钟(参见附注10)。
    5. 用100μlPBS洗涤细胞两次。
    6. 将细胞用100μl4%PFA在室温下固定15分钟
    7. 用100μlPBS洗涤细胞三次,每次5分钟。
    8. 用100μlDAPI溶液染色DNA 1分钟
    9. 用100μlPBS洗涤细胞三次。
    10. (可选,见注11)用100μlPyronin Y溶液染色RNA 15秒
    11. (可选)用100μlPBS洗涤细胞三次。
    12. 向每个孔中加入100μlPBS,并用96孔板密封膜密封孔。
    13. 使用IN Cell Analyzer 1000(图2)可视化金星信号并检测样品中的核灶。在400倍放大倍数下拍摄图像,自动优化曝光时间和硬件自动对焦。从每个井获得了25个不同领域的图像。 (见注12)


      图2. NBs的RNase敏感性筛选的图像。通过用Pyronin Y染色观察细胞RNA,并通过监测耐RNase抗性Cajal体的持续性(由COIL-维纳斯)和RNase敏感性散斑虫(由SFPQ-Venus标记)的消失。 Sam68核体是新鉴定的RNase敏感型NB。箭头表示核病灶。 DNA用DAPI(蓝色)染色。 Bar =10μm。

数据分析

通过使用IN Cell Analyzer 1000软件计数作为核的概述的DAPI阳性椭圆形区域来确定细胞数目(n> 100)。手工计数核心阳性细胞数。核糖核酸酶敏感性通过核糖核酸酶处理的细胞中的核聚焦阳性细胞数相对于没有核糖核酸酶处理的细胞中的数目的比例来测量。

笔记

  1. 该解决方案应在使用时准备。
  2. 尽管这里描述的方案是用于HeLa细胞的,但是它可能适用于其他培养的细胞系(例如HCT116,U2OS和NIH3T3)。这些细胞系可以从几个细胞库中心获得,例如 American Type Culture Collection
  3. 培养基应适当选择使用的细胞系。
  4. 表达定位于某些核病灶的金星标记蛋白的FLJ克隆的列表可在以下网站获得: http://hgpd.lifesciencedb.jp/sys_info/download.html 。维纳斯标记的人类cDNA产物的定位图像可以在人类基因和蛋白质数据库( HGPD)。原始的Gateway Entry克隆可从生物资源中心获得,国家技术与评估研究所(NBRC)。我们使用两种金星标记的蛋白作为对照:SFPQ-Venus(一种斑马鱼的标记,RNase敏感性NB)和COIL-Venus(Cajal体的标记,RNase抗性NB)。
  5. 该解决方案应在使用时准备。
  6. 可以根据所使用的细胞选择其它涂层试剂如阳离子聚合物(例如,聚-L-赖氨酸)或基质蛋白(例如,层粘连蛋白,纤连蛋白) 。
  7. 细胞通道数应少于5个。
  8. 应通过改变IT-LT1试剂的浓度来优化转染条件。
  9. 通过监测对照金星标记蛋白(注4)和Pyronin Y染色,应优化渗透条件。应对每种使用的细胞系优化吐温20的浓度。应该注意的是,用较高浓度吐温20的治疗常常导致金星信号在细胞中的消失
  10. 应通过监测对照金星标记蛋白(注4)和Pyronin Y染色优化RNA酶条件。我们总是比较两种具有和不具有RNase处理的样品用于一个cDNA克隆。
  11. 通过用Pyronin Y染色细胞RNA来确认RNA酶处理的效果。
  12. 为了对所选cDNA产物的定位位点的最终判断,应确认N-末端和C-末端金星标记的蛋白质,以一致地定位在相同的NB中。此外,相应内源蛋白的定位应通过用特异性抗体标记来确认

食谱

  1. 胶原蛋白溶液
    在0.05N HCl中稀释8.6μl/ml胶原蛋白
  2. 1M Tris-HCl,pH 7.4(1L)
    用超纯水稀释121.1克Tris,用盐酸调节pH至7.4
  3. 渗透缓冲液
    20mM Tris-HCl,pH 7.4
    5mM MgCl 2
    0.5 mM EGTA
    1x完全无EDTA蛋白酶抑制剂鸡尾酒
  4. 4%PFA
    向PBS中加入0.4 g PFA,并将体积调整为10 ml
    在55℃下剧烈振荡,直到完全溶解,并通过0.45μm过滤器滤出
  5. DAPI解决方案
    加入10毫克DAPI超纯水,调节体积为1毫升(10毫克/毫升DAPI原料)
    储存于-20°C
    使用前,稀释1μl10 mg/ml DAPI原液在10ml PBS(1μg/ml DAPI)中
  6. Pyronin Y解决方案
    加入3mg Pyronin Y超纯水,调节体积至10ml(1mM Pyronin Y库存)
    储存于-20°C
    使用前,用PBS(1μMPyronin Y)稀释1000倍

致谢

这个协议是从我们最近出版的作品(Mannen等人,2016年)改编而成。我们感谢国立先进工业科学技术研究所的Goshima,Kawamura和H. Mochizuki在制备金星标记的cDNA文库方面的支持。这项研究得到了日本教育,文化,体育,科学和技术部赠款的支持(26113002和26291001)。

参考文献

  1. Audas,TE,Jacob,MD和Lee,S。(2012)。通过核糖体间质非编码RNA固定核仁中的蛋白质。细胞 45(2):147-157。
  2. Biamonti,G.和Vourc'h,C.(2010)。 Cold Spring Harb Perspect Biol 2(6):a000695。
  3. Chen,LL and Carmichael,GG(2009)。  已更改在人胚胎干细胞中含有反向重复的mRNA的核保留:核非编码RNA的功能作用。细胞 35(4):467-478。
  4. Chujo,T.,Yamazaki,T。和Hirose,T。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26021608" target ="_ blank">建筑RNA(arcRNA):一类用作核体的支架的长非编码RNA。 Biochim Biophys Acta 1859(1):139-146。
  5. Clemson,CM,Hutchinson,JN,Sara,SA,Ensminger,AW,Fox,AH,Chess,A.and Lawrence,JB(2009)。  核非编码RNA的构建角色:NEAT1 RNA对于散斑结构是必不可少的。 em> 33(6):717-726。
  6. Fox,AH,Bond,CS and Lamond,AI(2005)。  Paraspeckles:一个新颖的核域。 Curr Biol 12(1):13-25。
  7. Hirose,T.和Goshima,N。(2015)。使用荧光标记的FLJ cDNA克隆文库的核体成分的基因组共定位筛选。方法Mol Biol 1262:155-163。
  8. Mannen,T.,Yamashita,S.,Tomita,K.,Goshima,N。和Hirose,T。(2016)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/27377249"target ="_ blank"> Sam68核体由两个由适配器HNRNPL连接的核糖核酸酶敏感亚结构组成。细胞生物 214 1):45-59。
  9. 毛泽东,YS,Zhang,B.和Spector,DL(2011)。核体的生物发生和功能。 趋势基因 27(8):295-306。
  10. Naganuma,T.,Nakagawa,S.,Tanigawa,A.,Sasaki,YF,Goshima,N。和Hirose,T。(2012)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/22960638"target ="_ blank">长非编码RNA的替代3'末端处理启动构建核paras。。。。。。。。。。。。。。。。。。。。。 20):4020-4034。
  11. Praserh,KV,Prasanth,SG,Xuan,Z.,Hearn,S.,Freier,SM,Bennett,CF,Zhang,MQ and Spector,DL(2005)。  通过RNA核保留调节基因表达。 Cell 123(2): 249-263。
  12. Sasaki,YT,Ideue,T.,Sano,M.,Mituyama,T.and Hirose,T。(2009)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov/pubmed/19188602"target ="_ blank"> MENepsilon /β非编码RNA对于核散斑的结构完整性至关重要。 Proc Natl Acad Sci USA 106(8): 2525-2530。
  13. Sleeman,JE和Trinkle-Mulcahy,L.(2014)。  。Curr Opin Cell Biol 28:76-83。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mannen, T. and Hirose, T. (2017). RNase Sensitivity Screening for Nuclear Bodies with RNA Scaffolds in Mammalian Cells. Bio-protocol 7(8): e2232. DOI: 10.21769/BioProtoc.2232.
  2. Mannen, T., Yamashita, S., Tomita, K., Goshima, N. and Hirose, T. (2016). The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL. J Cell Biol 214(1): 45-59.
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