Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences

Thomas D. Schaal; Tom Maniatis

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Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences

机译：前mRNA剪接增强子的选择和表征：新型SR蛋白特异性增强子序列的鉴定。

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Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers.

机译：剪接增强子是精确剪接位点识别和替代剪接控制所需的RNA序列。在这项研究中，我们使用了体外选择程序来鉴定和表征能够充当前mRNA剪接增强子的新型RNA序列。将随机的18个核苷酸的RNA序列插入到 Drosophila doublesex pre-mRNA增强子依赖性剪接底物的下游。然后通过在核提取物中进行多轮体外剪接，逆转录和剪接产物的选择性PCR扩增来选择功能性剪接增强子。所选剪接增强子的表征揭示了高度异质的序列，但是我们鉴定了六类重复的简并序列基序，长度为5至7个核苷酸，包括新型剪接增强子序列基序。在S100互补分析中对所选剪接增强子元件和其他增强子的分析导致鉴定了能够被富含特定丝氨酸/精氨酸（SR）的剪接因子（SC35、9G8和SF2 / ASF）激活的单个增强子。另外，在选择中分离的有效剪接增强子序列特异性结合20-kDa SR蛋白。该增强子序列与最近鉴定的可以用SRp20特异性抗体免疫沉淀的RNA-蛋白质加合物具有高度的序列同源性。我们得出结论，特定增强子可以激活不同类别的所选增强子，但是每个类别中都有相当大的序列简并性。结合以前的研究，此处给出的结果揭示了能够结合特异性SR蛋白和/或用作SR特异性剪接增强子的RNA序列范围广。 展开▼

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《Molecular and Cellular Biology》 |1999年第3期|共15页

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Thomas D. Schaal; Tom Maniatis;
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中图分类细胞生物学;

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专利

1. In vitro selection of exonic splicing enhancer sequences: identification of novel CD44 enhancers [J] . Gertrud Woerfel, Albrecht Bindereif Nucleic Acids Research . 2001,第15期

机译：外显子剪接增强子序列的体外选择：新型CD44增强子的鉴定

2. In vitro selection of exonic splicing enhancer sequences: identification of novel CD44 enhancers [J] . Albrecht Bindereif, Gertrud Woerfel Nucleic acids research . 2001,第15期

机译：外显子剪接增强子序列的体外选择：新型CD44增强子的鉴定

3. Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence. [J] . Marquis J, Meyer K, Angehrn L, Molecular therapy: the journal of the American Society of Gene Therapy . 2007,第8期

机译：脊髓性肌萎缩症：SMN2前mRNA剪接被带有剪接增强子序列的U7 snRNA衍生物纠正。

4. Joint Prediction of Branchpoint and Splice Acceptor Sites in pre-mRNA Transcripts Using Sequence to Sequence Networks [C] . Sanket Rajan Gupte, Rajgopal Srinivasan IEEE International Conference on Bioinformatics and Biomedicine . 2018

机译：使用序列到序列网络联合预测前mRNA转录本中的分支点和剪接受体位点

5. Identification and characterization of enhancer sequences in the 5'-flanking region of the fatty acid synthase gene responsible for in vivo rates of gene transcription. [D] . Rufo, Caterina Rosa. 2001

机译：脂肪酸合酶基因5'侧翼区中负责体内基因转录速率的增强子序列的鉴定和表征。

6. Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences [O] . Thomas D. Schaal, Tom Maniatis 1999

机译：前mRNA剪接增强子的选择和表征：新型SR蛋白特异性增强子序列的鉴定。

7. Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences [O] . Schaal, Thomas D., Maniatis, Tom 1999

机译：前mRNA剪接增强子的选择和表征：新型SR蛋白特异性增强子序列的鉴定。

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7. 保守性与特异性人源增强子序列识别及转录调控的研究 [A] . 张思佳 . 2019

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3. Enhanced pre-mRNA (RTM) trans splice molecules and their uses [P] . 外国专利： ES2579477T3 . 2016-08-11

机译：增强的pre-mRNA（RTM）跨剪接分子及其用途

4. Modified gene encoding a rna, protein, polypeptide or peptide, vector, methods for controlling the production of a protein, polypeptide or peptide from a gene, to modify a gene, to remove a 5'-cap structure and / or a 3'-pulley tail of a pre-mRNA or mRNA transcribed from a gene, for controlled production of a protein, polypeptide or peptide within a cell, of evolution and in vitro selection for the production of a ligand-bound RNA sequence of evolution and in vitro selection for the production of a ligand-dependent self-cleavable RNA sequence, synthetic DNA molecule, combination of ligand and modified gene, dna, and ligand-dependent self-cleaving synthetic RNA molecule [P] . 外国专利： BR0014632A . 2002-06-18

机译：编码RNA，蛋白质，多肽或肽的修饰基因，载体，控制基因中蛋白质，多肽或肽的产生，修饰基因，去除5'-帽结构和/或3'-的方法从基因转录的前mRNA或mRNA的滑轮尾，用于控制细胞内蛋白质，多肽或肽的产生，进化和体外选择，以产生配体结合的RNA进化和体外选择序列用于生产依赖配体的自切割RNA序列，合成DNA分子，配体和修饰基因的组合，dna和依赖配体的自切割合成RNA分子

5. isolated polypetide, composition, recombinant host cell, methods for producing the polypetide, a polypetide having cellulolytic enhancer activity, a precursor cell mutant, a protein and a fermentation product, transgenic plant, plant part or plant cell, double stranded inhibitor (rsrna), and methods for inhibiting expression of a polypeptide having cellulolytic enhancer activity in a cell, degrading or converting an isolated polypeptide, composition, recombinant host cell, methods for producing the polypetide, a polypetide having activity cellulolytic enhancer, a precursor cell mutant, a protein and a fermentation product, a transgenic plant, a plant part or a plant cell, a double stranded inhibitor (dsrna) molecule, and methods for inhibiting expression of a polypeptide having cellulolytic enhancer activity in a cell, degrade or convert r a cellulosic material and ferment a cellulosic material. [P] . 外国专利： BR112012006032A2 . 2015-09-08

机译：分离的多肽，组合物，重组宿主细胞，多肽的生产方法，具有纤维素分解增强活性的多肽，前体细胞突变体，蛋白质和发酵产物，转基因植物，植物部分或植物细胞，双链抑制剂（rsrna），抑制细胞中具有纤维素分解增强子活性的多肽的表达的方法和方法，降解或转化分离的多肽，组合物，重组宿主细胞，生产多肽的方法，具有纤维素分解增强子活性的多肽，前体细胞突变体，蛋白质和发酵产物，转基因植物，植物部分或植物细胞，双链抑制剂（dsrna）分子，以及抑制具有纤维素分解增强活性的多肽在细胞中表达，降解或转化纤维素物质并发酵的方法纤维素材料。

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Selection and Characterization of Pre-mRNA Splicing Enhancers: Identification of Novel SR Protein-Specific Enhancer Sequences

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