MECHANISM OF MEIOSIS-SPECIFIC RNA SPLICING IN YEAST

酿酒酵母MER1和MER2基因的产物需要 减数分裂过程中的重组和染色体分离。MER1基因 只在减数分裂细胞中转录。MER2基因转录于 营养细胞和减数分裂细胞，但转录本被剪接以产生 只有在减数分裂中才有功能信息。MER2转录本的剪接 需要MER1基因的产物。中国的长期目标是 提出的研究是对心力衰竭分子机制的理解 减数分裂特异的，依赖于MER1的RNA剪接。 MER2基因将在体外进行诱变，以鉴定顺式-β-内酰胺酶。 在剪接调控中起作用的作用序列。特别是， MER2 5‘拼接接头(GUUCGU)将更改为共识5’ 剪接位点序列(GUAUGU)。 野生型营养细胞剪接提取物(MER1-)和 来自在有丝分裂生长过程中表达MER1基因的工程细胞 (MER1+)将用于演示MER1依赖的MER2剪接 体外转录。将尝试确定MER1是否 蛋白质通过以下方式在剪接中发挥直接作用：(1)热激活 温度敏感的mer1突变体的提取物，(Ii)添加MER1 细菌中产生的蛋白质到MER1-提取物和/或(Iii)使用抗- 免疫耗竭MER1+提取物的MER1抗体。剪接体将是 分离并检测MER1蛋白的存在。此外, 将尝试证明MER1蛋白与 凝胶阻滞法测定MER2转录本；结合部位为 通过化学修饰/干扰实验绘制了图谱。 为了确定在MER2剪接中起作用的反式作用因子， 将分离出mer1突变体的第二位抑制因子。这些 实验将使用mer2：：URA3融合基因，其表达 依赖于剪接；因此，MER2转录本 可在不含尿嘧啶的培养基上选择剪接。人们希望， 可以分离出等位基因特异性抑制基因，这些基因将定义 其产物直接与MER1蛋白相互作用的基因。 携带无内含子MER2基因(CMER2)的Mer1菌株在 减数分裂，表明MER1需要剪接at的转录本 除了MER2，至少还有另外一个基因。为了鉴定MER1- 依赖基因，将筛选酵母基因组文库中的基因 当过度表达时，抑制了mer1 CMER2的减数分裂缺陷 菌株。另一种方法涉及prp26突变体，它 累积内含子(通过剪接释放)作为未降解的连接。 将从prp26mer1和prp26mer1减数分裂细胞中分离出秘书处，并 用于筛选酵母基因组文库。与内含子杂交的克隆 从MER1，而不是MER1，菌株将得到进一步的特征。

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.