Mechanism and Regulation of mRNA Export

mRNA输出的机制和调控

• 批准号: 8157188
• 负责人: RAVI DHAR
• 金额: $ 71.66万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157188
• 关键词: 14-3-3 Family; Cytoplasm; DNA Damage; DNA Repair; DNA damage checkpoint; Data; Homologous Gene; Mitotic; Pore Proteins; Regulation; Saccharomycetales; chromatin immunoprecipitation; mRNA Export; protein complex

The nuclear export of mRNA is mediated by proteins that are associated with it in the nucleus. Export factors are recruited to the RNA during transcriptional elongation. Some of the proteins associated with the mRNA, target the mRNP to the NPC for export out of the nucleus. In the cytoplasm the mRNP is disassembled, RNA is used for translation and the proteins are returned to the nucleus. Recently, using tandem affinity purification (TAP) of genomic tagged rae1, we identified several nuclear pore proteins that associate with Rae1 in fission yeast. We found S. pombe Nup189, Nup186, Nup184, Nup170, Nup146, Nup132, Nup124, Nup120, Nup107, Npp106, Nup97, Nup61, Nup45, Nup44, Nup40 and Nsp1 co-purified with TAP tagged Rae1. In budding yeast these above NPC proteins have been found to form several NPC-protein complexes, some involved with nuclear export and some with nuclear import. Our data would indicate that Rae1p appears to be associated with several NPC-complexes during the nucleocytoplasmic exchange process. Additionally, we found that Dss1p a DNA repair and mRNA export factor, along with mitotic activator Cdc25p, messenger RNA export/cell cycle factor Rae1p, 19 S proteasomal factors, and recombination protein Rhp51p (a Rad51p homolog) copurified with a DNA-damage checkpoint protein TAP tagged Rad24, a member of the 14-3-3 family of proteins . Using chromatin immunoprecipitation, we found that Dss1p recruited Rad24 and Rae1 to the double-strand break (DSB) sites. Furthermore, Cdc25 also recruited to the DSB site, and its recruitment was dependent on Dss1, Rad24, and the protein kinase Chk1. Following DSB, all nuclear Cdc25 was found to be chromatin-associated. We found that Dss1 and Rae1 have a DNA damage checkpoint function, and upon treatment with UV light a null strain of dss1 cells entered mitosis prematurely with indistinguishable timing from the null rad24 cells. Taken together, these results suggest that Dss1 plays a critical role in linking repair and checkpoint factors to damaged DNA sites by specifically recruiting Rad24 and Cdc25 to the DSBs. We have suggested that the sequestration of Cdc25 to DNA damage sites could provide a mechanism for S. pombe cells to arrest at G2/M boundary in response to DNA damage. The involvement of mRNA export factors in multiple cellular processes raises the possibility that mRNA export and the DNA-damage/ cell cycle processes may be linked.

mRNA 的核输出是由细胞核中与其相关的蛋白质介导的。在转录延伸过程中，输出因子被招募到 RNA 中。一些与 mRNA 相关的蛋白质将 mRNP 靶向 NPC，以便从细胞核中输出。在细胞质中，mRNP 被分解，RNA 用于翻译，蛋白质返回细胞核。最近，使用基因组标记 rae1 的串联亲和纯化 (TAP)，我们鉴定了裂殖酵母中与 Rae1 相关的几种核孔蛋白。我们发现粟酒裂殖酵母 Nup189、Nup186、Nup184、Nup170、Nup146、Nup132、Nup124、Nup120、Nup107、Npp106、Nup97、Nup61、Nup45、Nup44、Nup40 和 Nsp1 与 TAP 标记的 Rae1 共纯化。在芽殖酵母中，已发现这些上述 NPC 蛋白形成几种 NPC-蛋白复合物，一些与核输出有关，一些与核输入有关。我们的数据表明，Rae1p 似乎在核质交换过程中与多个 NPC 复合体相关。此外，我们发现 Dss1p 是一种 DNA 修复和 mRNA 输出因子，以及有丝分裂激活剂 Cdc25p、信使 RNA 输出/细胞周期因子 Rae1p、19 S 蛋白酶体因子和重组蛋白 Rhp51p（Rad51p 同源物）与 DNA 损伤检查点共纯化蛋白质 TAP 标记为 Rad24，是 14-3-3 蛋白质家族的成员。使用染色质免疫沉淀，我们发现 Dss1p 将 Rad24 和 Rae1 招募到双链断裂 (DSB) 位点。此外，Cdc25也招募到DSB位点，并且其招募依赖于Dss1、Rad24和蛋白激酶Chk1。 DSB 后，发现所有核 Cdc25 均与染色质相关。我们发现 Dss1 和 Rae1 具有 DNA 损伤检查点功能，并且在用紫外线处理后，dss1 细胞无效株过早进入有丝分裂，其时间与无效 rad24 细胞无法区分。总而言之，这些结果表明，Dss1 通过专门将 Rad24 和 Cdc25 招募到 DSB，在将修复和检查点因子与受损 DNA 位点连接起来方面发挥着关键作用。我们认为，Cdc25 与 DNA 损伤位点的隔离可以为粟酒裂殖酵母细胞响应 DNA 损伤而停滞在 G2/M 边界提供一种机制。 mRNA 输出因子在多个细胞过程中的参与提出了 mRNA 输出和 DNA 损伤/细胞周期过程可能存在关联的可能性。