Probing the specificity and activity of the metazoan Integrator complex

探讨后生动物整合复合体的特异性和活性

• 批准号: 10437741
• 负责人: Karen L Adelman
• 金额: $ 64.95万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10437741
• 关键词: Address; Amino Acids; Attenuated; Behavior; Binding; Biochemical; Biological; Bruck-de Lange syndrome; C-terminal; Cells; Chromatin; Code; Complex; Computer Analysis; Cues; Data; Development; Disease; Drosophila genus; Elongation Factor; Endoribonucleases; Enhancers; Explosion; Functional disorder; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Transcription; Growth; Hematological Disease; Hematopoiesis; Hematopoietic Neoplasms; Homeostasis; Human Development; In Vitro; Knowledge; Malignant Neoplasms; Malignant neoplasm of liver; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Modification; Molecular; Monitor; Mutation; Neurons; Output; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Plasmids; Polymerase; Property; Protein Dephosphorylation; Protein phosphatase; Proteins; RNA; RNA Polymerase II; RNA Splicing; RNA analysis; RNA chemical synthesis; Regulation; Regulator Genes; Reporter; Research; Role; Serine; Signal Transduction; Site; Small Nuclear RNA; Specificity; Stimulus; Structure; Time; Transcript; Transcription Elongation; Transcriptional Regulation; Untranslated RNA; Work; attenuation; base; cilium biogenesis; cohesin; developmental disease; endonuclease; experience; functional genomics; gene repression; human disease; improved; in vivo; insight; interest; lipid biosynthesis; malignant stomach neoplasm; migration; mutant; neurogenesis; promoter; protein complex; public health relevance; recruit; response; synergism; termination factor; tool; transcription factor; transcription termination; transcriptome sequencing

PROJECT SUMMARY: Precise control of gene expression in response to external signals and cues is essential for organismal development, growth and homeostasis. In metazoan cells, the regulated pausing of RNA polymerase II (RNAPII) in early transcript elongation is pivotal for defining gene output. However, the mechanisms that govern pausing and release of RNAPII into productive elongation remain poorly defined. Our work demonstrates that the Integrator complex (INT) targets paused RNAPII to attenuate messenger RNA (mRNA) expression. Moreover, we find that INT is a central regulator of gene expression in Drosophila cells, through its selective recruitment to specific gene promoters and subsequent INT-mediated termination of transcription. INT is known to be required for 3'-end formation of small nuclear RNAs involved in splicing (snRNAs), and has been implicated in cleavage and processing of other non-coding RNAs. Accordingly, the Integrator 11 subunit (IntS11) was found to possess an RNA endonuclease activity. Interestingly, INT was recently shown to also associate with RNAPII at protein- coding genes, however, our understanding of INT function on mRNAs is very limited. As described below, our preliminary data and proposed research will address fundamental, unanswered questions about gene regulation, and aim to elucidate the roles of INT in human disease. For example, paused RNAPII is very stable at some mRNA gene promoters, yet highly unstable at others. This diversity of behaviors for paused RNAPII has generated considerable interest concerning its underlying causes. In particular, the evidence for rapid turnover of paused RNAPII suggests the presence of a promoter-proximal termination factor. However, no such factor has been described in metazoan cells. Our work demonstrates that INT is this ‘missing’ termination factor and suggests a role for IntS11-mediated RNA cleavage in transcript termination. In addition, we discovered that INT recruits a protein phosphatase, PP2A, to RNAPII leading to de-phosphorylation of the RNAPII C-terminal domain (CTD). This finding is highly significant, since RNAPII CTD phosphorylation is involved in the release of paused RNAPII into productive mRNA elongation. The proposed work expands on this preliminary data. Aim 1 will determine how INT is targeted to specific gene promoters, pursuing in particular evidence for an interaction between INT and the cohesin complex. Notably, INT has already been shown to interact with cohesin in mammalian neurons, and implicated together with cohesin in Cornelia de Lange Syndrome. Aim 2 will probe the impact of the IntS11 RNA endonuclease activity on RNAPII elongation properties. Aim 3 will define the substrates and consequences of INT-mediated recruitment of the PP2A phosphatase to the early elongation complex. Critically, mutations in INT have been associated with a large number of diseases, with every one of the 14 subunits in the INT complex being implicated a pathophysiological state(s). Consequently, improving our understanding of INT activity will provide invaluable insights into mechanisms of diseases such as blood, hepatic and gastric cancers, and developmental disorders, including aberrant neuronal migration.

项目总结:

项目成果

Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination.

• DOI: 10.1016/j.molcel.2020.08.016
• 发表时间: 2020-10-15
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Huang KL;Jee D;Stein CB;Elrod ND;Henriques T;Mascibroda LG;Baillat D;Russell WK;Adelman K;Wagner EJ
• 通讯作者: Wagner EJ