論文 - 詳細
| RRC ID | 53419 |
|---|---|
| 著者 | Zhang H, Abraham N, Khan LA, Gobel V. |
| タイトル | RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic acid-based metabolites such as lipids. |
| ジャーナル | Nat Protoc |
| Abstract |
The field of metabolomics continues to catalog new compounds, but their functional analysis remains technically challenging, and roles beyond metabolism are largely unknown. Unbiased genetic/RNAi screens are powerful tools to identify the in vivo functions of protein-encoding genes, but not of nonproteinaceous compounds such as lipids. They can, however, identify the biosynthetic enzymes of these compounds-findings that are usually dismissed, as these typically synthesize multiple products. Here, we provide a method using follow-on biosynthetic pathway screens to identify the endpoint biosynthetic enzyme and thus the compound through which they act. The approach is based on the principle that all subsequently identified downstream biosynthetic enzymes contribute to the synthesis of at least this one end product. We describe how to systematically target lipid biosynthetic pathways; optimize targeting conditions; take advantage of pathway branchpoints; and validate results by genetic assays and biochemical analyses. This approach extends the power of unbiased genetic/RNAi screens to identify in vivo functions of non-nucleic acid-based metabolites beyond their metabolic roles. It will typically require several months to identify a metabolic end product by biosynthetic pathway screens, but this time will vary widely depending, among other factors, on the end product's location in the pathway, which determines the number of screens required for its identification. |
| 巻・号 | 10(5) |
| ページ | 681-700 |
| 公開日 | 2015-5-1 |
| DOI | 10.1038/nprot.2015.031 |
| PII | nprot.2015.031 |
| PMID | 25837419 |
| PMC | PMC5597045 |
| MeSH | Animals Biosynthetic Pathways* / genetics Caenorhabditis elegans* / genetics Caenorhabditis elegans* / metabolism Caenorhabditis elegans Proteins / genetics Caenorhabditis elegans Proteins / metabolism Enzymes / genetics Enzymes / metabolism Lipid Metabolism / physiology* Metabolomics / instrumentation Metabolomics / methods* Phospholipids / genetics Phospholipids / metabolism RNA Interference* Sphingolipids / genetics Sphingolipids / metabolism Workflow |
| IF | 10.419 |
| 引用数 | 6 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | X(Twitter) |
| 各媒体での言及数の合計 | 3 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
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