RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic acid–based metabolites such as lipids

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 non-proteinaceous 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.

Multilayered Reprogramming in Response to Persistent DNA Damage in C. elegans

“…SLs are highly conserved components of cell membranes having regulatory roles in growth control and aging in a wide range of organisms ( Cutler et al., 2014 ). SL works as an intermediate for the production of ceramide (Cer), a key product for the synthesis of glucosylceramide and sphingomyelin (SM) ( Zhang et al., 2015 ) ( Figure 4 A). Cer is produced from SM in UV- and IR-treated mammalian cells ( Zeidan et al., 2008 ), whereas an increased synthesis of SM from Cer is associated to accelerated development and aging ( Cutler et al., 2014 ).…”

“…Phosphatidic acid is dephosphorylated to yield DAG, which is converted into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which can be both intermediates for the formation of phosphatidylserine (PS). PS and phosphatidylinositol (PI) are generally synthesized from cytidine diphosphatediacylglycerol (CDP-DAG), substrate for the synthesis of phosphatidylglycerol (PG) and cardiolipin ( Zhang et al., 2015 ). Quantitative glycerophospholipids MS profiling, upon starvation and UV treatment, showed a change of the DAG downstream products, indicating a preferential direction in the phospholipid synthesis ( Figures 4 D and S4 ).…”

Sphingolipidomic Analysis of C. elegans reveals Development- and Environment-dependent Metabolic Features

“…The worm samples were thawed and homogenated firstly by a sonicator (Fisher Scientific, Model 505 sonic dismembrator, Fair Lawn, NJ, USA), and then freeze-dried (FreeZone 4.5 Freeze Dryer, Labconco, Kansas City, MO, USA) for 24 hrs. The extraction of SLs from C. elegans was carried out according to a modified Bligh and Dyer's method 37, 38. Briefly, 30 mg of worms were added with 2 ml of methanol (MeOH) and 1 ml of chloroform (CHCl 3 ), vortexed for 1 min, and incubated in a water bath (TW12, Julabo, Allentown, PA) at 48°C for 24 hrs.…”

Proteotoxic stress disrupts epithelial integrity by inducing MTOR sequestration and autophagy overactivation

“…To investigate possible roles of Kelch repeat-containing proteins in modulating intestinal morphogenesis, we carried out RNAi knockdown of 11 C. elegans proteins containing Kelch repeats (based on the SMART database) using worms expressing the lumenal membrane marker ACT-5::GFP. We identified that the previously uncharacterized gene C53A5.6 shows fully penetrant L1 larval lethality with severe intestinal morphogenesis defects upon standard RNAi treatment [ 31 ] ( Figure 1 and Table S1). The protein encoded by C53A5.6 is distinct from other Kelch repeat proteins owing to the presence of an N-terminal RING domain; therefore, we named this gene rike-1 ( RI NG- and Ke lch-containing protein).…”