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2015 ; 10
(5
): 681-700
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RNAi-based biosynthetic pathway screens to identify in vivo functions of
non-nucleic acid-based metabolites such as lipids
#MMPMID25837419
Zhang H
; Abraham N
; Khan LA
; Gobel V
Nat Protoc
2015[May]; 10
(5
): 681-700
PMID25837419
show ga
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.
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