SignalSilence? TRIM33 siRNA I

• 产品编号：CST-13487S      品牌：CST       原厂货号：13487S
• 产品分类：DNA和RNA纯化 > RNA干扰 > RNAi Oligos > siRNA
• 应用分类：

描述：

来自Cell Signaling Technology (CST)的SignalSilence^® TRIM33 siRNA I可以帮助研究者通过RNA干扰特异性地抑制TRIM33的表达，这种方法可以通过将双链RNA分子传递到细胞内从而使基因表达有选择的沉默。来自CST的所有的SignalSilence^® siRNA产品都是经过内部严格检测的，并且通过Western blot 分析证明确实能够减少目的蛋白的表达。通过三苯甲基分析每个碱基以监测寡核苷酸的合成，确保合适的配对效率。随后寡核苷酸通过亲和固相萃取法纯化。退火的RNA双链通过质谱分析来证实其精确的组成。每一批产品都通过质谱分析与前面的产品进行比较，来保证不同批次之间的最大一致性。CST推荐使用100 nM SignalSilence^® TRIM33 siRNA I 进行转染，48到72小时后对细胞进行裂解。转染步骤按照转染试剂说明书提供的步骤进行。遇到任何使用方面的问题，请随时联系CST。每小瓶可供100次转染，每次转染量相当于在转染24孔板时，每孔总体积为300μl培养基中siRNA的终浓度为100nM。转录中介因子1 (TIF1)家族代表一类包含多个组蛋白结合结构与的蛋白。人体中，该家族由四个蛋白组成，TIF1α/TRIM24, TIF1β/TRIM28/KAP1, TIF1γ/TRIM33/Ectodermin, 和TIF1δ/TRIM66,其特点是含有一个RING结构，两个B 盒子，一个卷曲螺旋结构域和一个羧基端PHD finger和bromodomain组成的氨基末端tripartite motif (TRIM)结构域(1)。除了类似的结构，这些蛋白在转录调控中表现了多种功能。TIF1α功能是配体依赖性核受体辅助调节因子，最近认为它涉及调节p53的稳定性(2)。TIF1β是N-COR1辅阻遏物复合物和NURD核小体重塑复合物的固有成分（3），用作Kruppel-associated box（KRAB）的锌指转录因子的辅阻遏物(4)。不仅如此，TIF1β作为异染色质蛋白HP1的辅助因子促进异染色质介导的基因沉默(5). TIF1δ的表达限制在睾丸中，并证明能和HP1γ相互作用(6)。广泛表达的核蛋白质TRIM33不与任一HP1家庭成员或染色质重构/修饰复合物相互作用。与此相反，TRIM33在配体TGF-β超家族主导的的信号级联反应中发挥了重要作用(7-9)。研究指出TRIM33和Smad4竞争结合磷酸化的受体Smad2/3，TRIM33-Smad2/3和Smad4-Smad2/3复合物在TGF-β依赖的造血细胞命运决定过程中功能互补(9)。但是其他研究证明了TRIM33抑制了TGF-β超家族的信号(7-8,10)。实际上，敲除小鼠的Trim33会导致Nodal信号上升最终胚胎致死(10)。理论上，TRIM33作为一个E3泛素连接酶，会促进Smad4的单泛素化，这个修饰将削弱它和磷酸化Smad2结合的能力(8)。这个负调控机制进一步由TRIM33破坏竞争Smad复合物和TGF-β反应基因的启动子/增强子区域的结合能力而证实的，这个过程中TRIM33和表观遗传学标记组蛋白H3结合，组蛋白H3能够激活TRIM33对Smad4的单泛素化能力(11).与TRIM33调节不同血细胞系发育的能力一致，TRIM33的表观遗传学沉默导致的表达缺失会引起慢性粒细胞白血病的症状(12)。

1. Meroni, G. and Diez-Roux, G. (2005) Bioessays 27, 1147-57.
2. Jain, A.K. and Barton, M.C. (2009) Cell Cycle 8, 3668-74.
3. Underhill, C. et al. (2000) J Biol Chem 275, 40463-70.
4. Schultz, D.C. et al. (2001) Genes Dev 15, 428-43.
5. Groner, A.C. et al. (2010) PLoS Genet 6, e1000869.
6. Khetchoumian, K. et al. (2004) J Biol Chem 279, 48329-41.
7. Dupont, S. et al. (2005) Cell 121, 87-99.
8. Dupont, S. et al. (2009) Cell 136, 123-35.
9. He, W. et al. (2006) Cell 125, 929-41.
10. Morsut, L. et al. (2010) Development 137, 2571-8.
11. Agricola, E. et al. (2011) Mol Cell 43, 85-96.
12. Aucagne, R. et al. (2011) J Clin Invest 121, 2361-70.

原厂资料：

Description

SignalSilence^® TRIM33 siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit TRIM33 expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence^® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

Quality Control

Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.

Directions for Use

CST recommends transfection with 100 nM SignalSilence^® TRIM33 siRNA I 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use.

Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.

Background

The transcriptional intermediary factor 1 (TIF1) family represents a group of proteins with multiple histone-binding domains. In humans, this family comprises four proteins, TIF1α/TRIM24, TIF1β/TRIM28/KAP1, TIF1γ/TRIM33/Ectodermin, and TIF1δ/TRIM66, which are characterized by an amino-terminal tripartite motif (TRIM) domain consisting of a RING domain, two B boxes, a coiled-coil domain, and a carboxy-terminal PHD finger and bromodomain (1). Despite their similar overall structure, these proteins have diverse roles in transcriptional regulation. TIF1α functions as a ligand-dependent nuclear receptor coregulator and more recently has been implicated in regulating p53 stability (2). TIF1β is an intrinsic component of the N-CoR1 corepressor complex and the NuRD nucleosome-remodeling complex (3) and functions as a corepressor for Kruppel-associated box (KRAB) zinc-finger transcription factors (4). Furthermore, TIF1β promotes heterochromatin-mediated gene silencing formation by serving as a cofactor for heterochromatin protein HP1 (5). TIF1δ expression is restricted to the testis and has been shown to interact with HP1γ (6).

In contrast, the ubiquitous nuclear protein TRIM33 does not interact with either HP1 family members or chromatin-remodeling/modifying complexes. Rather, TRIM33 plays a pivotal role in signaling cascades driven by the TGF-β superfamily of ligands (7-9). A research study suggests that TRIM33 and Smad4 compete for binding to receptor phosphorylated Smad2/3 and that TRIM33-Smad2/3 and Smad4-Smad2/3 complexes complement one another in the TGF-β-dependent control of hematopoietic cell fate (9). Other studies, however, demonstrate that TRIM33 functions to repress signal relay by the TGF-β superfamily (7-8,10). Indeed, knockout of murine Trim33 results in embryonic lethality due to upregulated Nodal signaling (10). Mechanistically, TRIM33 functions as an E3-ubiquitin ligase and promotes monoubiquitination of Smad4, a modification that impairs its ability to associate with phospho-Smad2 (8). This negative regulatory mechanism is further substantiated by the discovery that TRIM33 disrupts transcriptionally competent Smad complexes on the promoter/enhancer regions of TGF-β-responsive genes by associating with specific epigenetic marks on histone H3, which is a requirement for activating TRIM33's monoubiquitin ligase activity toward Smad4 (11). In line with the ability of TRIM33 to regulate the development of different blood cell lineages, it was shown that loss of TRIM33 expression due to epigenetic silencing of its promoter contributes to the pathogenesis of chronic myelomonocytic leukemia (12).

1. Meroni, G. and Diez-Roux, G. (2005) Bioessays 27, 1147-57.
2. Jain, A.K. and Barton, M.C. (2009) Cell Cycle 8, 3668-74.
3. Underhill, C. et al. (2000) J Biol Chem 275, 40463-70.
4. Schultz, D.C. et al. (2001) Genes Dev 15, 428-43.
5. Groner, A.C. et al. (2010) PLoS Genet 6, e1000869.
6. Khetchoumian, K. et al. (2004) J Biol Chem 279, 48329-41.
7. Dupont, S. et al. (2005) Cell 121, 87-99.
8. Dupont, S. et al. (2009) Cell 136, 123-35.
9. He, W. et al. (2006) Cell 125, 929-41.
10. Morsut, L. et al. (2010) Development 137, 2571-8.
11. Agricola, E. et al. (2011) Mol Cell 43, 85-96.
12. Aucagne, R. et al. (2011) J Clin Invest 121, 2361-70.

注意事项：

Storage: TRIM33 siRNA I is supplied in RNAse-free water.
Aliquot and store at -20ºC.