RNA LARIATS, DEBRANCHING ENZYME, AND RETROVIRAL REPLICATION

RNA 套索、脱支酶和逆转录病毒复制

• 批准号: 7957826
• 负责人: THOMAS M MENEES
• 金额: $ 0.33万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7957826
• 关键词: Affect; Affinity; Alleles; Biology; California; Cell Survival; Computer Retrieval of Information on Scientific Projects Database; Data; Development; Elements; Enzymes; Exonuclease; Funding; Fungal Genome; Goals; Grant; HIV-1; Human; Institution; Integration Host Factors; Introns; Mass Spectrum Analysis; Methods; Phenotype; Preparation; Process; Proteins; RNA; RNA Splicing; Research; Research Personnel; Resources; Retroviridae; Retrovirus-like Element; Reverse Transcription; Ribonucleases; Role; Source; Staging; United States National Institutes of Health; Virus-like particle; Western Blotting; Yeasts; 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 goal of the Menees research group has been 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. A yeast strain containing a tandem affinity tagged (TAP-tagged) DBR1 allele as its only source of Dbr1p was used to purify Dbr1p-TAP. This yeast strain is wild-type for the processes that depend on Dbr1p: RNA lariat debranching and Ty element transposition. Mass spectrometric analysis revealed that Ty1 proteins were found to specifically copurify with Dbr1p-TAP, raising the possibility that Dbr1p is present within Ty1 virus-like particles (VLPs). Such an association would support a specific host factor role for Dbr1p in Ty1 replication. However, contrasting data were obtained with partially purified Ty1 VLP preparations: Dbr1p was detected by western blotting but not by mass spectrometry. Using more purified Ty1 VLPs obtained by an immunoaffinity method, Dbr1p is still not detected by mass spectrometry. We are distinguishing between the following two possibilities: (1) Dbr1p may be present in VLPs but mass spectrometry may not be able to reveal its presence or (2) the TAP-tagged version of Dbr1p spuriously associates with VLPs. The development of an immunoaffinity method to purify Ty1 VLPs opens up a new window for studying Ty1 host factors. Such studies will be helpful in assessing the roles of host factors for human retroviruses.

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研究小组的一个目标是了解DBR1P如何成为逆转录病毒和类似逆转录病毒元素的宿主因素。 实现此目标的一种方法是确定酵母DBR1P的相互作用伙伴。 包含带有串联亲和力的酵母菌菌株，标记为DBR1P的唯一来源用于净化DBR1P-TAP。 对于依赖于DBR1P的过程，该酵母菌菌株是野生型：RNA Lariat分解和TY元素转座。 质谱分析表明，发现TY1蛋白与DBR1P-TAP特别相交，从而提高了DBR1P存在于TY1病毒样颗粒（VLP）中的可能性。 这种关联将支持DBR1P在TY1复制中的特定宿主因子角色。 但是，通过部分纯化的TY1 VLP制剂获得对比数据：通过蛋白质印迹检测到DBR1P，但不能通过质谱法检测到。 使用通过免疫亲和力方法获得的更纯化的TY1 VLP，DBR1P仍未通过质谱检测到DBR1P。 我们正在区分以下两种可能性：（1）DBR1P可能存在于VLP中，但是质谱可能无法揭示其存在，或者（2）DBR1P的TAPAGED版本dbr1p伪造的与VLPS相关联。 纯化TY1 VLP的免疫亲和力方法的开发为研究TY1宿主因子的新窗口打开了新窗口。 此类研究将有助于评估人类逆转录病毒的宿主因素的作用。