MECHANISM OF MEIOSIS-SPECIFIC RNA SPLICING IN YEAST

酵母减数分裂特异性 RNA 剪接机制

• 批准号: 3306816
• 负责人: GLENNA ROEDER
• 金额: $ 19.25万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306816
• 关键词: RNA splicing; Saccharomyces cerevisiae; binding proteins; crosslink; fungal genetics; fusion gene; gene deletion mutation; gene expression; genetic library; genetic mapping; genetic regulatory element; genetic transcription; immunochemistry; meiosis; molecular cloning; mutant; nucleic acid sequence; point mutation; spliceosomes; temperature sensitive mutant; transcription factor

The products of the MER1 and MER2 genes of S.cerevisiae are required for recombination and chromosome segregation during meiosis. The MER1 gene is transcribed only in meiotic cells. The MER2 gene is transcribed in both vegetative and meiotic cells but the transcript is spliced to yield a functional message only in meiosis. Splicing of the MER2 transcript requires the product of the MER1 gene. The long term goal of the proposed research is an understanding of the molecular mechanism of meiosis-specific, MER1-dependent RNA splicing. The MER2 gene will be mutagenized in vitro in order to identify cis- acting sequences that play a role in splicing regulation. In particular, the MER2 5' splice junction (GUUCGU) will be changed to the consensus 5' splice site sequence (GUAUGU). Splicing extracts prepared from wild-type vegetative cells (MER1-) and from cells engineered to express the MER1 gene during mitotic growth (MER1+) will be used to demonstrate MER1-dependent splicing of the MER2 transcript in vitro. Attempts will be made to determine whether the MER1 protein plays a direct role in splicing by (i) heat activation of extracts from a temperature-sensitive mer1 mutant, (ii) adding MER1 protein produced in bacteria to MER1- extracts and/or (iii) using anti- MER1 antibodies to immunodeplete MER1+ extracts. Spliceosomes will be isolated and examined for the presence of the MER1 protein. In addition, attempts will be made to demonstrate binding of the MER1 protein to the MER2 transcript by gel retardation assays; the binding site will be mapped by chemical modification/interference experiments. To identify trans-acting factors that play a role in MER2 splicing, second-site suppressors of mer1 mutants will be isolated. These experiments will employ a mer2::URA3 fusion gene whose expression depends on splicing; thus, mutants in which the MER2 transcript is spliced can be selected on medium lacking uracil. It is hoped that allele-specific suppressors can be isolated and that these will define genes whose products interact directly with the MER1 protein. mer1 strains carrying an intronless MER2 gene (cMER2) are defective in meiosis, suggesting that MER1 is required to splice the transcript of at least one other gene in addition to MER2. In order to identify MER1- dependent genes, a yeast genomic library will be screened for genes which, when overexpressed, suppress the meiotic defect of mer1 CMER2 strains. An alternative approach involves the prp26 mutant, which accumulates introns (released by splicing) as undegraded lariats. Lariats will be isolated from prp26 MER1 and prp26mer1 meiotic cells and used to screen a yeast genomic library. Clones that hybridize to introns from MER1, but not mer1, strains will be characterized further.

酿酒酵母的MER 1和MER 2基因的产物是 减数分裂过程中的重组和染色体分离。MER 1基因 只在减数分裂细胞中转录。MER 2基因转录于 营养细胞和减数分裂细胞，但转录本被剪接， 只有在减数分裂中才有功能的信息。MER 2转录本的剪接 需要MER 1基因的产物。的长期目标 建议的研究是对分子机制的理解， 减数分裂特异性，MER 1依赖性RNA剪接。 MER 2基因将在体外诱变，以鉴定顺式- 在剪接调节中起作用的作用序列。特别是， MER 2 5'剪接点（GUUCGU）将被改变为共有5' 剪接位点序列（GUAUGU）。 从野生型营养细胞（MER 1-）制备的剪接提取物和 来自在有丝分裂生长期间表达MER 1基因的细胞 (MER1+）将用于证明MER 2的MER 1依赖性剪接 体外转录。将尝试确定MER 1是否 蛋白质在剪接中起直接作用，通过（i）热激活 来自温度敏感性mer 1突变体的提取物，（ii）加入MER 1 细菌中产生的蛋白质与MER 1-提取物和/或（iii）使用抗- MER 1抗体免疫耗竭MER 1+提取物。剪接体将是 分离并检查MER 1蛋白的存在。此外，本发明还提供了一种方法， 将尝试证明MER 1蛋白与 通过凝胶阻滞测定MER 2转录物;结合位点将是 通过化学修饰/干扰实验绘制。 为了鉴定在MER 2剪接中起作用的反式作用因子， 将分离MER 1突变体的第二位点抑制子。这些 实验将使用mer 2：：URA 3融合基因，其表达 取决于剪接;因此，突变体中的MER 2转录本是 可以在缺乏尿嘧啶的培养基上选择拼接的。人们希望 可以分离出等位基因特异性抑制子，这些抑制子将定义 其产物直接与MER 1蛋白相互作用的基因。 携带无内含子MER 2基因（cMER 2）的mer 1菌株在以下方面存在缺陷： 减数分裂，表明MER 1是拼接at转录本所必需的 除了MER 2之外，还存在至少一种其它基因。为了鉴定MER 1- 依赖基因，酵母基因组文库将筛选基因 当其过表达时，抑制mer 1 CMER 2的减数分裂缺陷 菌株另一种方法涉及prp 26突变体， 积累内含子（通过剪接释放）作为未降解的内含子。 将从prp 26 MER 1和prp 26 mer 1减数分裂细胞中分离出落叶松， 用于筛选酵母基因组文库。与内含子杂交的克隆 来自MER 1而不是mer 1的菌株将被进一步表征。