MOUSE MINUTES: A GENETIC STUDY OF RIBOSOMAL PROTEINS

小鼠分钟：核糖体蛋白的遗传学研究

• 批准号: 7105069
• 负责人: Thomas M. Glaser
• 金额: $ 30.22万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105069
• 关键词: cellular pathology; complementary DNA; embryonic stem cell; gene expression; gene expression profiling; gene mutation; genetic disorder; genetic models; genetic transduction; genetically modified animals; hematopoiesis; laboratory mouse; molecular pathology; phenotype; polysomes; protein structure function; retinal ganglion; ribosomal proteins; tissue /cell culture

DESCRIPTION (provided by applicant): The spontaneous mouse mutation Bst (belly spot and tail) causes midventral spotting, vertebral anomalies (tail kinks), preaxial polydactyly, a reduction in retinal ganglion cell (RGC) number, optic nerve aplasia, colobomata, and subretinal neovascularization (Rice et al. 1997; Smith et al. 2000). Bst homozygotes die before implantation. We have identified Bst as a deletion within Rpl24, the gene encoding the large subunit ribosomal protein L24. The mutation causes aberrant splicing in 80% of transcripts, leading to truncation of the L24 polypeptide. Mouse BAC and human cDNA transgenes correct the Bst/+ phenotype. Almost nothing is known about ribosomal protein (RP) gene mutations in vertebrates. In Drosophila, numerous ribosomal gene mutations, termed Minutes, are dispersed across the genome (Lambertsson 1998). They are homozygous lethal and have similar heterozygous phenotypes consisting of smaller, thinner bristles and profoundly delayed development (Schultz 1929). Their effects are not additive when combined. Minutes were instrumental in pioneering studies that defined concepts of cell autonomy, clonal lineage and developmental compartmentation, as Minute cells compete poorly with wild type clones in somatic mosaics (Simpson and Morata 1981). In humans, Diamond-Blackfan anemia (hereditary red blood cell aplasia with variable congenital anomalies) is caused by mutations in RPS19, which encodes a small subunit riboprotein (Willig et al. 1999). In view of the universal cellular requirement for ribosomes, the tissue specificity of mouse Rpl24 and human RPS19 phenotypes is puzzling. Our findings suggest that riboprotein defects may underlie common human malformation syndromes that have a suspected genetic etiology but no single target locus (Lalani et al. 2003). Fibroblasts from Bst/+ embryos grow slower than wild-type MEFs, remain longer in G1 phase, and exhibit decreased rates of protein synthesis. Although complete phenotypic surveys are lacking, the majority of tissues compensate for this cellular growth defect, so that organogenesis proceeds and Bst/+ mice approach normal adult size. While extraribosomal functions for L24 are possible, unique Bst phenotypes can also be explained by a dyschronic mechanism, in tissues such as the retina where proliferation and the sequence of cell fate determination are relatively uncoupled. In this proposal, we aim to: (1) complete characterization of Bst molecular, cellular and tissue developmental effects; (2) investigate the selective growth disadvantage of Rpl24 deficient cells in ROSA26 lacZ <-> Bst/+ blastocyst chimeras and somatic mosaics, as predicted by classical Minute studies; and (3) define the spectrum of riboprotein phenotypes by comparing Bst/+ mice to selected knockout mice with other RP mutations, including several that are available as targeted ES cells.

描述（由申请人提供）：小鼠自发突变 Bst（腹斑和尾部）导致腹中点斑点、椎骨异常（尾部扭结）、轴前多指畸形、视网膜神经节细胞 (RGC) 数量减少、视神经发育不全、缺损和视网膜下新生血管形成（Rice 等人，1997 年；Smith 等人，2000 年）。 Bst 纯合子在植入前死亡。我们已将 Bst 鉴定为 Rpl24（编码大亚基核糖体蛋白 L24 的基因）内的缺失。该突变导致 80% 的转录物出现异常剪接，导致 L24 多肽截短。小鼠 BAC 和人类 cDNA 转基因可纠正 Bst/+ 表型。关于脊椎动物核糖体蛋白（RP）基因突变几乎一无所知。在果蝇中，许多核糖体基因突变（称为“分钟”）分散在整个基因组中（Lambertsson 1998）。它们是纯合致死的，并具有相似的杂合表型，包括更小、更薄的刚毛和严重延迟的发育（Schultz 1929）。它们的效果在组合时不会相加。 Minute 在定义细胞自主性、克隆谱系和发育区划概念的开创性研究中发挥了重要作用，因为 Minute 细胞与体细胞嵌合体中的野生型克隆竞争较差（Simpson 和 Morata 1981）。在人类中，Diamond-Blackfan 贫血（具有多种先天性异常的遗传性红细胞再生障碍性贫血）是由编码小亚基核糖蛋白的 RPS19 突变引起的（Willig 等，1999）。鉴于细胞对核糖体的普遍需求，小鼠 Rpl24 和人类 RPS19 表型的组织特异性令人费解。我们的研究结果表明，核糖蛋白缺陷可能是常见人类畸形综合征的基础，这些畸形综合征具有可疑的遗传病因，但没有单一的靶基因座（Lalani et al. 2003）。 来自 Bst/+ 胚胎的成纤维细胞比野生型 MEF 生长得更慢，在 G1 期停留的时间更长，并且蛋白质合成率降低。尽管缺乏完整的表型调查，但大多数组织弥补了这种细胞生长缺陷，因此器官发生继续进行，并且 Bst/+ 小鼠接近正常成年体型。虽然 L24 的核糖体外功能是可能的，但独特的 Bst 表型也可以用一种不同步的机制来解释，在视网膜等组织中，增殖和细胞命运决定的序列相对不相关。 在本提案中，我们的目标是：（1）完整表征 Bst 分子、细胞和组织发育效应； (2) 研究 Rpl24 缺陷细胞在 ROSA26 lacZ <-> Bst/+ 囊胚嵌合体和体细胞嵌合体中的选择性生长劣势，正如经典 Minute 研究所预测的那样； (3) 通过将 Bst/+ 小鼠与选定的具有其他 RP 突变的敲除小鼠（包括几种可用作靶向 ES 细胞的突变）进行比较，定义核糖蛋白表型谱。