RNA LARIATS, DEBRANCHING ENZYME, AND RETROVIRAL REPLICATION

RNA 套索、脱支酶和逆转录病毒复制

• 批准号: 7602184
• 负责人: THOMAS M MENEES
• 金额: $ 0.17万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7602184
• 关键词: Affect; Affinity; Alleles; California; Cell Survival; Complex; Computer Retrieval of Information on Scientific Projects Database; Elements; Endoribonucleases; Enzymes; Exonuclease; Funding; Goals; Grant; HIV-1; Human; Institution; Integration Host Factors; Introns; Mass Spectrum Analysis; Methods; Modeling; Object Attachment; Pancreatic ribonuclease; Peptides; Phenotype; Process; Proteins; Protocols documentation; RNA; RNA Splicing; Research; Research Personnel; Resources; Retroviridae; Retrovirus-like Element; Reverse Transcription; Ribonucleases; Source; Staging; United States National Institutes of Health; Western Blotting; Work; Yeasts; design; lariat debranching enzyme

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Debranching enzyme, Dbr1p, is an RNase that promotes intron RNA turnover after splicing. The enzyme is a phosphoesterase that cuts the 2'-5' bond at intron RNA lariat branch points, allowing exonucleases to access the RNA. Loss of Dbr1p function results in accumulation of intron lariat RNAs, which has a minimal effect on yeast cell viability. A second phenotype associated with loss of Dbr1p function in yeast is decreased replication of the retrovirus-like elements Ty1 and Ty3, with reverse transcription being the specific replication stage affected. This latter phenotype is not understood, however a group in California has shown that the retrovirus HIV-1 requires the human version of the Dbr1 enzyme for reverse transcription. A major goal of the Menees research group is to understand how Dbr1p acts as a host factor for retroviruses and retrovirus-like elements. One approach for achieving this goal is to determine the interacting partners for yeast Dbr1p. If Ty element proteins or other known host factors for Ty replication are identified as partners of Dbr1p, better models for how Dbr1p is acting in retroviral replication can be developed. Any identified interactions will be studied further. A yeast strain containing a tandem affinity tagged (TAP-tagged) DBR1 allele as its only source of Dbr1p has been obtained. This yeast strain is wild type for the processes that depend on Dbr1p: RNA lariat debranching and Ty element transposition. Thus, biologically relevant interactions for Dbr1p should also occur with Dbr1p-TAP. Dbr1p-TAP has been purified from yeast cultures according to established protocols designed to preserve protein complexes. Western blots of purified Dbr1p-TAP indicate that the purification method is working on this TAP-tagged protein. Purified Dbr1p-TAP (and any interacting proteins) will be proteolytically digested and the peptides will analyzed by mass spectrometry. Results will be compared to a negative control to assess potential interacting partners for Dbr1p.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 DBR1P脱节酶是一种RNase，可在剪接后促进内含子RNA转换。 该酶是一种磷酸酯酶，可在内含子RNA lar raiat分支点上切割2'-5'键，从而使外切核酸酶进入RNA。 DBR1P功能的丧失会导致内含子幼虫RNA的积累，这对酵母细胞活力的影响最小。 与酵母中DBR1P功能丧失相关的第二种表型减少了类似逆转录病毒的元素Ty1和Ty3，其逆转录是受到特定的复制阶段的影响。 后一种表型尚不清楚，但是加利福尼亚州的一组表明，逆转录病毒HIV-1需要DBR1酶的人类版本才能进行逆转录。 Menees研究小组的一个主要目标是了解DBR1P如何成为逆转录病毒和类似逆转录病毒的元素的宿主因素。 实现此目标的一种方法是确定酵母DBR1P的相互作用伙伴。 如果将复制的TY元素蛋白或其他已知宿主因子确定为DBR1P的伙伴，则可以开发出更好的DBR1P在逆转录病毒复制中起作用的模型。 任何已确定的互动都将进一步研究。 已获得包含串联亲和力的酵母菌菌株，标记为DBR1的DBR1等位基因是其唯一的DBR1P来源。 对于依赖于DBR1P的过程，该酵母菌菌株是野生类型：RNA lar脱离分支和TY元件换位。 因此，DBR1P的生物学相关相互作用也应与DBR1P-TAP发生。 根据旨在保留蛋白质复合物的既定方案，DBR1P-TAP已从酵母培养物中纯化。 纯化的DBR1P-TAP的Western印迹表明，纯化方法正在使用这种粘液性蛋白质。 纯化的DBR1P-TAP（以及任何相互作用的蛋白质）将被蛋白水解消化，肽将通过质谱法分析。 将结果与阴性对照进行比较，以评估DBR1P的潜在相互作用伴侣。