The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion i...The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.展开更多
Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons re...Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.展开更多
Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach i...Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach is proposed for designing artificial trans-acting sRNAs.By engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence.By applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin biosynthesis.This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B.subtilis and E.coli but also enhanced our understanding of trans-acting sRNAs.展开更多
目的探究敲减Rho鸟苷三磷酸酶激活蛋白30(Rho GTPase-activating protein 30,ARHGAP30)后,宫颈癌Siha细胞增殖及凋亡的变化。方法设计特异性shARHGAP30引物并连接pLKO.1载体,转化到大肠杆菌感受态细胞中,再与慢病毒辅助质粒转入HEK-293...目的探究敲减Rho鸟苷三磷酸酶激活蛋白30(Rho GTPase-activating protein 30,ARHGAP30)后,宫颈癌Siha细胞增殖及凋亡的变化。方法设计特异性shARHGAP30引物并连接pLKO.1载体,转化到大肠杆菌感受态细胞中,再与慢病毒辅助质粒转入HEK-293T细胞,收集细胞上清获得的病毒过滤后感染Siha细胞,RT-qPCR和Western blot检测敲减效率,以及转染后Bax及Bcl-2的表达变化;CCK-8法检测敲减后细胞的增殖水平。结果成功构建敲减ARHGAP30基因的慢病毒质粒,并建立Siha稳转细胞,ARHGAP30在Siha细胞中的转录和翻译减少(P<0.01),Bax/Bcl-2明显降低(P<0.01),凋亡减少,细胞增殖水平升高(P<0.01)。结论ARHGAP30参与Siha细胞的增殖与凋亡,调控ARHGAP30基因或将干扰宫颈癌的发生和发展。展开更多
<i><span style="font-family:Verdana;">Centella asiatica </span></i><span style="font-family:""><span style="font-family:Verdana;">(L.) is one of t...<i><span style="font-family:Verdana;">Centella asiatica </span></i><span style="font-family:""><span style="font-family:Verdana;">(L.) is one of the most valuable medicinal plants since preh</span><span style="font-family:Verdana;">istoric times. The pharmaceutical importance of this herb is due to the accumulation of large quantities of pentacyclic triterpenoid saponins, collectively known as centelloids synthesized by the isoprenoid biosynthesis</span><span style="font-family:Verdana;"> path</span><span style="font-family:Verdana;">way. Biosynthesis of triterpenoid in the plants proceeds via either of the tw</span><span style="font-family:Verdana;">o pathways, viz. Mevalonate (MVA) pathway (in the cytosol) or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (in plastid). In </span><i><span style="font-family:Verdana;">Centella</span></i><span style="font-family:Verdana;">, the pathway leading to the accumulation of triterpenoid is still not known or elucidated. Thus, to know whether the MVA or MEP pathway or a cross-talk between the pathway leads to the biosynthesis of triterpenoid, silencing the key regulatory gene using RNAi tool, of each of the pathway and then analyze a metabolite is an efficient approach. The key regulatory enzyme of the MVA pathway </span><i><span style="font-family:Verdana;">i.e</span></i><span style="font-family:Verdana;">. 3-</span><i><span style="font-family:Verdana;">Hydroxy</span></i><span style="font-family:Verdana;">-3-</span><i><span style="font-family:Verdana;">methylglutaryl-coenzyme A reductase</span></i><span style="font-family:Verdana;"> (</span><i><span style="font-family:Verdana;">HMGR</span></i><span style="font-family:Verdana;">) has already been successfully silenced using RNAi tool</span></span><span style="font-family:Verdana;"> <a href="#ref1">[1]</a></span><span style="font-family:""><span style="font-family:Verdana;">. In the present study, the 1-</span><i><span style="font-family:Verdana;">deoxy-D-xylulose</span></i><span style="font-family:Verdana;">-5-</span><i><span style="font-family:Verdana;">phosphate reductoisomerase</span></i><span style="font-family:Verdana;"> (</span><i><span style="font-family:Verdana;">DXR</span></i><span style="font-family:Verdana;">) a key regulatory enzyme in MEP pathway is silenced. The RNAi-</span><i><span style="font-family:Verdana;">DXR</span></i><span style="font-family:Verdana;"> construct in pHANNIBAL vector was cloned into a binary vector pART27 and subsequently transformed into </span><i><span style="font-family:Verdana;">Agrobacterium</span></i><span style="font-family:Verdana;"> strain AGL1. The transient analysis of the RNAi-</span><i><span style="font-family:Verdana;">CaDXR</span></i><span style="font-family:Verdana;"> using semi-quantitative RT-PCR confirmed the silencing of the endogenous DXR gene in </span><i><span style="font-family:Verdana;">Nicotiana</span></i><span style="font-family:Verdana;"> and further confirmed in </span><i><span style="font-family:Verdana;">Centella asiatica</span></i><span style="font-family:Verdana;">. The present study is the first step aimed to delineate the MEP pathway using RNAi silencing approach to elucidate its role in the accumulation of triterpenoid in this important medicinal plant.展开更多
In small cell lung cancer cells, various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. The contribution of the Gq/11-PLC-β cascade to the mitogenic effects in SCLC cells is well esta...In small cell lung cancer cells, various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. The contribution of the Gq/11-PLC-β cascade to the mitogenic effects in SCLC cells is well established, but the relevance of G12/13 signaling is less explored. While in prostate and breast cancer, G12/13 activation has been shown previously to promote invasiveness without being involved in cellular proliferation, previous data from our group indicate anti-proliferative effects of G12/13 knockdown in small cell lung cancer (SCLC) cells. To further investigate the role of G12/13-dependent signaling in lung tumor cells, we employed shRNA-mediated targeting of Gα12, Gα13, or both, in SCLC and NSCLC cell lines. Lentiviral expression of shRNAs resulted in specific Gα12 and Gα13 knockdown. Of note, upon single knockdown of one family member, no counter-upregulation of the other one was observed. Interestingly, inhibition of proliferation was cell line dependent. In cell lines where knock-down led to antiproliferation, single knockdown of either Gα12 or Gα13 was sufficient to impair proliferation and double knockdown of Gα12 and Gα13 tended not to further increase anti-proliferative effects. Likewise, when single knockdown was insufficient for an inhibition of proliferation, no effects were observed in double knockdowns. Taken together, these findings indicate that both Gα12 and Gα13 affect cellular proliferation individually and interference with one family member is sufficient for anti-tumor effects.展开更多
RNA interference(RNAi)using small interfering RNA(siRNA)has shown potential as a therapeutic option for the treatment of arthritis by silencing specific genes.However,siRNA delivery faces several challenges,including ...RNA interference(RNAi)using small interfering RNA(siRNA)has shown potential as a therapeutic option for the treatment of arthritis by silencing specific genes.However,siRNA delivery faces several challenges,including stability,targeting,off-target effects,endosomal escape,immune response activation,intravascular degradation,and renal clearance.A variety of nanotherapeutics like lipidic nanoparticles,liposomes,polymeric nanoparticles,and solid lipid nanoparticles have been developed to improve siRNA cellular uptake,protect it from degradation,and enhance its therapeutic efficacy.Researchers are also investigating chemical modifications and bioconjugation to reduce its immunogenicity.This review discusses the potential of siRNA nanotherapeutics as a therapeutic option for various immune-mediated diseases,including rheumatoid arthritis,osteoarthritis,etc.siRNA nanotherapeutics have shown an upsurge of interest and the future looks promising for such interdisciplinary approach-based modalities that combine the principles of molecular biology,nanotechnology,and formulation sciences.展开更多
Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes a...Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3’-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition.3’-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5’-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.展开更多
基金supported by the National Natural Science Foundation of China,No.81460193
文摘The micro RNA（mi RNA） let-7 was one of the first mi RNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1（MKP1） is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase（MAPK） and c-Jun N-terminal kinase（JNK） expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.
基金supported by the National Natural Science Foundation of China,No.81372698
文摘Pitx3 is strongly associated with the phenotype, differentiation, and survival of dopaminergic neurons. The relationship between Pitx3 and glial cell line-derived neurotrophic factor（GDNF） in dopaminergic neurons remains poorly understood. The present investigation sought to construct and screen a lentivirus expression plasmid carrying a rat Pitx3 short hairpin（sh）RNA and to assess the impact of Pitx3 gene knockdown on GDNF transcriptional activity in MES23.5 dopaminergic neurons. Three pairs of interference sequences were designed and separately ligated into GV102 expression vectors. These recombinant plasmids were transfected into MES23.5 cells and western blot assays were performed to detect Pitx3 protein expression. Finally, the most effective Pitx3 sh RNA and a dual-luciferase reporter gene plasmid carrying the GDNF promoter region（GDNF-luciferase） were cotransfected into MES23.5 cells. Sequencing showed that the synthesized sequences were identical to the three Pitx3 interference sequences. Inverted fluorescence microscopy revealed that the lentivirus expression plasmids carrying Pitx3-sh RNA had 40-50% transfection efficiency. Western blot assay confirmed that the corresponding Pitx3 of the third knockdown sequence had the lowest expression level. Dual-luciferase reporter gene results showed that the GDNF transcriptional activity in dopaminergic cells cotransfected with both plasmids was decreased compared with those transfected with GDNF-luciferase alone. Together, the results showed that the designed Pitx3-sh RNA interference sequence decreased Pitx3 protein expression, which decreased GDNF transcriptional activity.
基金supported by the National Natural Science Foundation of China (31970085)the National Key Research and Development Program of China (2021YFC2100800)the Jiangsu Province Natural Science Fund for Distinguished Young Scholars (BK20200025).
文摘Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach is proposed for designing artificial trans-acting sRNAs.By engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence.By applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin biosynthesis.This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B.subtilis and E.coli but also enhanced our understanding of trans-acting sRNAs.
文摘目的探究敲减Rho鸟苷三磷酸酶激活蛋白30(Rho GTPase-activating protein 30,ARHGAP30)后,宫颈癌Siha细胞增殖及凋亡的变化。方法设计特异性shARHGAP30引物并连接pLKO.1载体,转化到大肠杆菌感受态细胞中,再与慢病毒辅助质粒转入HEK-293T细胞,收集细胞上清获得的病毒过滤后感染Siha细胞,RT-qPCR和Western blot检测敲减效率,以及转染后Bax及Bcl-2的表达变化;CCK-8法检测敲减后细胞的增殖水平。结果成功构建敲减ARHGAP30基因的慢病毒质粒,并建立Siha稳转细胞,ARHGAP30在Siha细胞中的转录和翻译减少(P<0.01),Bax/Bcl-2明显降低(P<0.01),凋亡减少,细胞增殖水平升高(P<0.01)。结论ARHGAP30参与Siha细胞的增殖与凋亡,调控ARHGAP30基因或将干扰宫颈癌的发生和发展。
文摘<i><span style="font-family:Verdana;">Centella asiatica </span></i><span style="font-family:""><span style="font-family:Verdana;">(L.) is one of the most valuable medicinal plants since preh</span><span style="font-family:Verdana;">istoric times. The pharmaceutical importance of this herb is due to the accumulation of large quantities of pentacyclic triterpenoid saponins, collectively known as centelloids synthesized by the isoprenoid biosynthesis</span><span style="font-family:Verdana;"> path</span><span style="font-family:Verdana;">way. Biosynthesis of triterpenoid in the plants proceeds via either of the tw</span><span style="font-family:Verdana;">o pathways, viz. Mevalonate (MVA) pathway (in the cytosol) or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (in plastid). In </span><i><span style="font-family:Verdana;">Centella</span></i><span style="font-family:Verdana;">, the pathway leading to the accumulation of triterpenoid is still not known or elucidated. Thus, to know whether the MVA or MEP pathway or a cross-talk between the pathway leads to the biosynthesis of triterpenoid, silencing the key regulatory gene using RNAi tool, of each of the pathway and then analyze a metabolite is an efficient approach. The key regulatory enzyme of the MVA pathway </span><i><span style="font-family:Verdana;">i.e</span></i><span style="font-family:Verdana;">. 3-</span><i><span style="font-family:Verdana;">Hydroxy</span></i><span style="font-family:Verdana;">-3-</span><i><span style="font-family:Verdana;">methylglutaryl-coenzyme A reductase</span></i><span style="font-family:Verdana;"> (</span><i><span style="font-family:Verdana;">HMGR</span></i><span style="font-family:Verdana;">) has already been successfully silenced using RNAi tool</span></span><span style="font-family:Verdana;"> <a href="#ref1">[1]</a></span><span style="font-family:""><span style="font-family:Verdana;">. In the present study, the 1-</span><i><span style="font-family:Verdana;">deoxy-D-xylulose</span></i><span style="font-family:Verdana;">-5-</span><i><span style="font-family:Verdana;">phosphate reductoisomerase</span></i><span style="font-family:Verdana;"> (</span><i><span style="font-family:Verdana;">DXR</span></i><span style="font-family:Verdana;">) a key regulatory enzyme in MEP pathway is silenced. The RNAi-</span><i><span style="font-family:Verdana;">DXR</span></i><span style="font-family:Verdana;"> construct in pHANNIBAL vector was cloned into a binary vector pART27 and subsequently transformed into </span><i><span style="font-family:Verdana;">Agrobacterium</span></i><span style="font-family:Verdana;"> strain AGL1. The transient analysis of the RNAi-</span><i><span style="font-family:Verdana;">CaDXR</span></i><span style="font-family:Verdana;"> using semi-quantitative RT-PCR confirmed the silencing of the endogenous DXR gene in </span><i><span style="font-family:Verdana;">Nicotiana</span></i><span style="font-family:Verdana;"> and further confirmed in </span><i><span style="font-family:Verdana;">Centella asiatica</span></i><span style="font-family:Verdana;">. The present study is the first step aimed to delineate the MEP pathway using RNAi silencing approach to elucidate its role in the accumulation of triterpenoid in this important medicinal plant.
文摘In small cell lung cancer cells, various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. The contribution of the Gq/11-PLC-β cascade to the mitogenic effects in SCLC cells is well established, but the relevance of G12/13 signaling is less explored. While in prostate and breast cancer, G12/13 activation has been shown previously to promote invasiveness without being involved in cellular proliferation, previous data from our group indicate anti-proliferative effects of G12/13 knockdown in small cell lung cancer (SCLC) cells. To further investigate the role of G12/13-dependent signaling in lung tumor cells, we employed shRNA-mediated targeting of Gα12, Gα13, or both, in SCLC and NSCLC cell lines. Lentiviral expression of shRNAs resulted in specific Gα12 and Gα13 knockdown. Of note, upon single knockdown of one family member, no counter-upregulation of the other one was observed. Interestingly, inhibition of proliferation was cell line dependent. In cell lines where knock-down led to antiproliferation, single knockdown of either Gα12 or Gα13 was sufficient to impair proliferation and double knockdown of Gα12 and Gα13 tended not to further increase anti-proliferative effects. Likewise, when single knockdown was insufficient for an inhibition of proliferation, no effects were observed in double knockdowns. Taken together, these findings indicate that both Gα12 and Gα13 affect cellular proliferation individually and interference with one family member is sufficient for anti-tumor effects.
文摘RNA interference(RNAi)using small interfering RNA(siRNA)has shown potential as a therapeutic option for the treatment of arthritis by silencing specific genes.However,siRNA delivery faces several challenges,including stability,targeting,off-target effects,endosomal escape,immune response activation,intravascular degradation,and renal clearance.A variety of nanotherapeutics like lipidic nanoparticles,liposomes,polymeric nanoparticles,and solid lipid nanoparticles have been developed to improve siRNA cellular uptake,protect it from degradation,and enhance its therapeutic efficacy.Researchers are also investigating chemical modifications and bioconjugation to reduce its immunogenicity.This review discusses the potential of siRNA nanotherapeutics as a therapeutic option for various immune-mediated diseases,including rheumatoid arthritis,osteoarthritis,etc.siRNA nanotherapeutics have shown an upsurge of interest and the future looks promising for such interdisciplinary approach-based modalities that combine the principles of molecular biology,nanotechnology,and formulation sciences.
文摘Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. In Drosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform. In situ hybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of all dADAR mRNA transcripts belong to the truncated class in embryos. 3’-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition.3’-UTR sequences for the truncated isoform have been determined from diverse Drosophila species and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in every Drosophila species studied, extending over a period spanning about 40 x 106 years, implying a conserved function. Previous work has shown that a dADAR protein isoform binds to the evolutionarily conserved rnp-4f pre-mRNA stem-loop located in the 5’-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncated dADAR protein isoform in Drosophila embryonic development, which is very likely evolutionarily conserved.
