siRNA对多头绒泡菌丝氨酸/精氨酸蛋白激酶表达的沉默

SR蛋白激酶（serine/arginine protein-specific kinase,SRPK）是一类特异磷酸化剪接因子SR蛋白的激酶家族,可调节SR蛋白的选择型剪接,影响SR蛋白在核内的分布与定位,在pre-mRNA剪接调控上具有重要的作用。目前对于多头绒泡菌SRPK的功能仍不清楚。为了进一步研究PSRPK的功能,设计可转录小干扰RNA（siRNA）的寡核苷酸序列含U6启动子的载体pSIREN-RetroQ定向连接,构建了真核表达质粒pSIREN-PSRPK-1,pSIREN-PSRPK-2,pSIREN-PSRPK-3,pSIREN-PSRPK-4,pSIREN-PSRPK-5及对照质粒pSIREN-PSRPK-Neg。把编码PSRPK基因的cDNA与红色荧光蛋白表达载体pDsRed-N1重组,构建了表达PSRPK-红色荧光融合蛋白的表达质粒pP-SRPK-DsRed。将真核表达质粒和质粒pPSRPK-DsRed共转染HEK293细胞。转染后72h通过倒置荧光显微镜观察,结果表明,pSIREN-PSRPK-2和pSIREN-PSRPK-5可以有效沉默红色荧光融合蛋白的表达;进一步采用RT-PCR和Northern点杂交分析表明,pSIREN-PSRPK-2和pSIREN-PSRPK-5对红色荧光融合蛋白mRNA表达具有明显的抑制作用与倒置荧光显微镜下观察到的结果一致。这为进一步研究多头绒泡菌SRPK的功能奠定了实验基础。     

hhlim基因表达产物的亚细胞定位及其在细胞肥大中的意义

hhlim是从胎儿心脏中新近分离和克隆得到的与心脏发生相关的基因,其表达产物作为转录因子参与多种基因的转录调控和细胞的发育与分化过程.用细胞转染方法将外源性hhlim基因导入处于分化过程中的小鼠成肌细胞C2C12,发现该基因强制性表达可使C2C12细胞体积明显增大.RT-PCR和蛋白质印迹结果表明,hhlim促细胞肥大与诱导α-肌动蛋白(α-actin)过表达及重新启动胚胎基因BNP表达有关.用绿色荧光蛋白-hhlim融合蛋白表达载体转染处于分化过程的C2C12细胞,发现转染不同时间,表达产物的亚细胞定位发生动态变化,表现为先在胞核和胞质中均有分布,而后定位于胞质的变化规律.应用免疫共沉淀证实,hhlim在胞质中以与α-actin 相互缔合的方式存在.     

PPARγ基因真核表达载体的构建及其在乳腺癌MDA—MB-231细胞中的表达

目的：构建以绿色荧光蛋白（GFP）为报告基因的重组表达质粒pEGFP—C1—PPARγ,观察小鼠PPARγ基因在MDA-MB-231细胞中的表达及定位。方法：采用克隆和亚克隆技术构建小鼠PPARγ基因真核表达载体,脂质体Lip2000介导转染MDA—MB-231细胞,real—time PCR和western—blot验证其mRNA和蛋白的表达,荧光显微镜观察该基因亚细胞定位。结果：酶切和测序结果证实重组质粒含有PPAIh编码区序列且插入方向正确,转染后观察该基因亚细胞定位于胞核,胞质有弥散分布。结论：成功构建了小鼠PPARγ基因真核表达载体,该基因在MDA—MB-231细胞中成功表达,PPARγ基因主要集中表达于胞核。     

丝氨酸/精氨酸蛋白特异激酶SRPK2在小鼠睾丸组织中的表达及意义

目的通过研究丝氨酸/精氨酸蛋白特异激酶2(serine/arginine-rich protein specific kinase 2,SRPK2)基因mRNA及其编码蛋白产物在小鼠睾丸组织中的表达特征,探讨该基因在精子发生过程中的作用及意义。方法分别采用半定量逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹杂交(Western blotting)分析该基因mRNA及蛋白产物在小鼠多种组织中的表达;利用实时定量PCR(real-time quantitative PCR)分析SRPK2 mRNA在不同发育阶段小鼠睾丸组织中的差异表达;应用免疫组织化学染色和间接免疫荧光技术观察SRPK2蛋白在小鼠曲精小管中的细胞定位和生精细胞内的亚细胞定位。结果半定量RT-PCR和Western blotting分析显示SRPK2 mRNA和蛋白在小鼠睾丸组织中均大量表达;实时定量PCR分析发现SRPK2 mRNA在5周及8周龄雄性小鼠睾丸组织中显著表达,具有明显的阶段特异性表达特征。免疫组织化学染色结果表明SRPK2蛋白阳性着色主要位于曲精小管中的长形精子细胞核;间接免疫荧光分析显示SRPK2蛋白定位于长形精子细胞核表面。结论 SRPK2基因在小鼠睾丸组织中大量表达,并且具有显著的阶段特异性表达特征和明确的细胞核定位,极有可能在小鼠精子发生的变态成形期参与mRNA前体分子的剪接过程,其作用机制值得进一步深入研究。     

PPARγ基因真核表达载体的构建及其在乳腺癌MDA-MB-231细胞中的表达

目的:构建以绿色荧光蛋白(GFP)为报告基因的重组表达质粒pEGFP-C1-PPARγ,观察小鼠PPARγ基因在MDA-MB-231细胞中的表达及定位.方法:采用克隆和亚克隆技术构建小鼠PPARγ基因真核表达栽体,脂质体Lip2000介导转染MDA-MB-231细胞,real-time PCR和western-blot验证其mRNA和蛋白的表达,荧光显微镜观察该基因亚细胞定位.结果:酶切和测序结果证实重组质粒含有PPARγ编码区序列且插入方向正确,转染后观察该基因亚细胞定位于胞核,胞质有弥散分布.结论:成功构建了小鼠PPARγ基因真核表达载体,该基因在MDA-MB-231细胞中成功表达,PPARγ基因主要集中表达于胞核.     

人DJ-1蛋白在NIH3T3细胞中的表达与定位

采用增强型绿色荧光蛋白（EGFP）示踪的方法,研究人DJ-1蛋白在真核细胞中的 定位及其对刺激的反应. 将克隆在pGEX-KG上的DJ-1亚克隆到载体pEGFP-C2上,对 阳性克隆进行PCR、酶切和测序鉴定,用脂质体转染NIH3T3细胞;并用荧光显微镜观察 pEGFP-DJ-1在细胞内的定位以及在血清刺激时的移位;探讨在氧化应激时DJ-1对细胞的保护作用,以及其在细胞内定位的变化. 重组质粒在NIH3T3细胞中得到高效表达,绿色荧光弥散分布于胞质、胞核中,但以胞质居多;血清刺激后,细胞中的绿色荧光从胞质移位到胞核;在200～600 μmol/L H₂O₂刺激下,DJ-1能有效保护细胞抵抗氧化应激,并且也能从胞质移位到胞核.上述研究结果为进一步研究DJ-1蛋白的功能提供了一个重要依据.     

绿色荧光蛋白与人肾脏NaDC3融合基因的构建及其在肾小管上皮细胞中的定位

本采用RT-PCR方法从人的正常肾组织中扩增出hNaDC3基因全长cDNA序列,采用基因重组技术将hNaDC3基因和绿色荧光蛋白基因融合,通过真核表达质粒—脂质体介导,导入LLC-PKl细胞系,激光共聚焦显微镜动态观察GFP—hNaDC3的定位情况。结果显示PKGFP-C3空白质粒转染的LLC—PKl细胞表达了GFP,GFP在胞内分布以核浆为主,呈均匀致密的细颗粒荧光,胞核染色强,核仁荧光稀少,界线清楚。而pKGFP-C3-hNaDC3转染细胞株的绿色荧光蛋白,转染后第1天混合分布于细胞浆和细胞膜,以粗颗粒荧光为主,两界限不清楚,胞浆中可见未染色的圆形空洞,未见胞核染色;第3天主要聚集于细胞膜,核周的胞浆中可见粗颗粒荧光物质,胞浆和胞膜界线清楚,胞核亦未见绿色荧光蛋白;第5天清晰聚集于细胞膜,细胞膜连接至网状。因此hNaDC3蛋白在细胞质中生成后,定位于细胞膜并稳定表达。     

布鲁氏菌分泌蛋白BspJ亚细胞定位分析

[目的]对布鲁氏菌分泌蛋白BspJ进行亚细胞定位分析,为深入探索该分泌蛋白的功能奠定基础。[方法]使用生物信息学方法对分泌蛋白BspJ的核酸和氨基酸序列进行生物信息学分析;利用常规PCR方法扩增BspJ目的基因并克隆到p MD19-T载体;构建真核表达载体p DsRed2-C1-BspJ并转染HEK293T细胞,制作细胞爬片后进行激光共聚焦显微镜分析;利用RT-PCR方法分析HEK293T转染细胞中BspJ转录情况。[结果]生物信息学分析结果显示,BspJ基因ORF全长是531 bp,共编码177个氨基酸,BspJ蛋白无跨膜结构,有7个抗原决定簇,具有核定位信号(NLS)和核输出信号(NES),据此推测BspJ蛋白亚细胞定位可能具有核—胞浆穿梭过程; PCR方法扩增出BspJ基因;成功构建p DsRed2-C1-BspJ重组质粒;激光共聚焦分析显示BspJ蛋白定位于宿主细胞核及核周围; RT-PCR分析显示BspJ基因于宿主细胞中进行转录。[结论]布鲁氏菌分泌蛋白BspJ分布于宿主细胞核及核周围,可能具有胞核—胞质穿梭过程。     

人1型酪蛋白激酶的真核表达及其在肿瘤细胞中的定位

目的:构建带FLAG标签的人1型酪蛋白激酶(CK1)基因的真核表达载体,获得其表达产物,并研究该激酶在骨肉瘤U2OS、乳腺癌ZR-75-1、肝癌HepG2等多种肿瘤细胞中的表达及定位情况。方法:应用PCR技术从人乳腺文库中扩增人CK1基因的全长编码区,将其克隆到带FLAG标签的pCMV-Tag2B载体中;将重组质粒转染骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞,以SDS-PAGE和Western印迹鉴定表达情况;细胞免疫荧光观察FLAG-CK1质粒在骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞中的细胞定位。结果:双酶切和测序结果显示FLAG-CK1真核表达质粒构建成功;SDS-PAGE和Western印迹结果表明,FLAG-CK1转染骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞后成功表达;细胞免疫荧光实验显示,CK1在骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞的胞核和胞质中均有分布,且胞核信号强于胞质。结论:构建了CK1的真核表达载体,且FLAG-CK1能在不同肿瘤细胞系的细胞核和细胞质中表达,为进一步研究CK1对细胞的调控奠定了实验基础。     

过氧化物酶蛋白1 真核表达载体构建及在Hela 细胞的表达与定位

目的:构建过氧化物酶蛋白1(PRDX 1)的真核表达载体,观察其在Hela细胞内表达及定位。方法:提取BALB/c小鼠肝脏组织总RNA,通过RT-PCR方法扩增得到小鼠PRDX 1编码序列,酶切后克隆至pcDNA3-myc载体。重组质粒通过PCR、酶切、测序证明构建正确后经脂质体转染Hela细胞,然后利用Western blot和荧光显微镜技术观察该融合蛋白在细胞内表达及定位。结果:经鉴定证明重组质粒构建正确;Western blot实验显示,该质粒能够在Hela细胞中特异表达;免疫荧光试验显示,蛋白产物分布在胞浆和胞核,证明该蛋白在细胞内高表达。结论:成功构建带有myc标签的PRDX 1真核表达载体,该质粒能够在哺乳细胞中特异表达并且外源性PRDX 1蛋白分布在Hela细胞胞浆胞核内,为深入研究PRDX 1蛋白在细胞内相关生物学研究奠定了基础。     

Localization of serine kinases, SRPK1 (SFRSK1) and SRPK2 (SFRSK2), specific for the SR family of splicing factors in mouse and human chromosomes

The serine- and arginine-rich (SR) splicing factors play an important role in both constitutive and alternative pre-mRNA splicing, and the functions of these splicing factors are regulated by phosphorylation. We have previously characterized SRPK1 (SFRSK1) and SRPK2 (SFRSK2), which are highly specific protein kinases for the SR family of splicing factors. Here we report the chromosomal localization of the mouse and human genes for both kinases. SRPK1 probes detected two loci that were mapped to mouse Chromosomes 17 and X using The Jackson Laboratory interspecific backcross DNA panel, and SRPK2 probes identified a single locus on mouse Chromosome 5. Using a somatic cell hybrid mapping panel and by fluorescence in situ hybridization, SRPK1 and SRPK2 were respectively mapped to human chromosomes 6p21.2-p21.3 (a region of conserved synteny to mouse Chromosome 17) and 7q22-q31.1 (a region of conserved synteny to mouse Chromosome 5). In addition, we also found multiple SRPK-related sequences on other human chromosomes, one of which appears to correspond to a SRPK2 pseudogene on human chromosome 8.     

SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells

Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH₂ terminus, and a recent study showed that this NH₂-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.     

Regulated cellular partitioning of SR protein-specific kinases in mammalian cells

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.     

Phosphorylation mechanism and structure of serine-arginine protein kinases

The splicing of mRNA requires a group of essential factors known as SR proteins, which participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition motifs and a C-terminal domain rich in Arg-Ser repeats (RS domain). SR proteins are phosphorylated at numerous serines in the RS domain by the SR-specific protein kinase (SRPK) family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events. Although SR proteins are polyphosphorylated in vivo, the mechanism underlying this complex reaction has only been recently elucidated. Human alternative splicing factor [serine/arginine-rich splicing factor 1 (SRSF1)], a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a post-translational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10-12 serines in the N-terminal region of the RS domain (RS1), using a mechanism that incorporates sequential, C-terminal to N-terminal phosphorylation and several processive steps. SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound to a docking groove in the large lobe of the kinase domain. Upon subsequent rounds of phosphorylation, this N-terminal segment translocates into the active site, and a β-strand in RNA recognition motif 2 unfolds and occupies the docking groove. These studies indicate that efficient regiospecific phosphorylation of SRSF1 is the result of a contoured binding cavity in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain, and a highly directional processing mechanism.     

The N-terminal fragment from caspase-cleaved serine/arginine protein-specific kinase2 (SRPK2) translocates into the nucleus and promotes apoptosis

SRPK2 belongs to a family of serine/arginine (SR) protein-specific kinases (SRPKs), which phosphorylate SR domain-containing proteins in the nuclear speckles and mediate the pre-mRNA splicing. Previous studies have shown that SRPK2 plays a pivotal role in cell proliferation and apoptosis. However, how SRPK2 is regulated during the apoptosis is unclear. Here, we show that SRPK2 is cleaved by caspases at Asp-139 and -403 residues. Its N terminus cleaved product translocates into the nucleus and promotes VP16-induced apoptosis. Akt phosphorylation of SRPK2 prevents its apoptotic cleavage by caspases. 14-3-3β, the binding partner of Akt-phosphorylated SRPK2, further protects it from degradation. Hence, our results suggest that the N-terminal domain of SRPK2 cleaved by caspases translocates into the nucleus, where it promotes chromatin condensation and apoptotic cell death.     

SRPK1 and LBR protein kinases show identical substrate specificities

Arginine/serine protein kinases constitute a novel class of enzymes that can modify arginine/serine (RS) dipeptide motifs. SR splicing factors that are essential for pre-mRNA splicing and the lamin B receptor (LBR), an integral protein of the inner nuclear membrane, are among the best characterized proteins that contain RS domains. Two SR Protein-specific Kinases, SRPK1 and SRPK2, have been shown to phosphorylate specifically the RS motifs of the SR family of splicing factors and play an important role in regulating both the spliceosome assembly and their intranuclear distribution, whereas an LBR-associated kinase, that specifically phosphorylates a stretch of RS repeats located at the NH2-terminal region of LBR, has been recently purified and characterized from turkey erythrocyte nuclear envelopes. Using synthetic peptides representing different regions of LBR and recombinant proteins produced in bacteria we now demonstrate that SRPK1 modifies LBR with similar kinetics and on the same sites as the LBR kinase, that are also phosphorylated in vivo. These data provide significant evidence for a new role of SRPK1 in addition to that of pre-mRNA splicing.     

Evidence for a role of Sky1p-mediated phosphorylation in 3' splice site recognition involving both Prp8 and Prp17/Slu4

The SRPK family of kinases is specific for RS domain-containing splicing factors and known to play a critical role in protein-protein interaction and intracellular distribution of their substrates in both yeast and mammalian cells. However, the function of these kinases in pre-mRNA splicing remains unclear. Here we report that SKY1, a SRPK family member in Saccharomyces cerevisiae, genetically interacts with PRP8 and PRP17/SLU4, both of which are involved in splice site selection during pre-mRNA splicing. Prp8 is essential for splicing and is known to interact with both 5' and 3' splice sites in the spliceosomal catalytic center, whereas Prp17/Slu4 is nonessential and is required only for efficient recognition of the 3' splice site. Interestingly, deletion of SKY1 was synthetically lethal with all prp17 mutants tested, but only with specific prp8 alleles in a domain implicated in governing fidelity of 3'AG recognition. Indeed, deletion of SKY1 specifically suppressed 3'AG mutations in ACT1-CUP1 splicing reporters. These results suggest for the first time that 3' AG recognition may be subject to phosphorylation regulation by Sky1p during pre-mRNA splicing.