CoLoC-seq probes the global topology of organelle transcriptomes

Abstract

Proper RNA localisation is essential for physiological gene expression. Various kinds of genome-wide approaches permit to comprehensively profile subcellular transcriptomes. Among them, cell fractionation methods, that couple RNase treatment of isolated organelles to the sequencing of protected transcripts, remain most widely used, mainly because they do not require genetic modification of the studied system and can be easily implemented in any cells or tissues, including in non-model species. However, they suffer from numerous false-positives since incompletely digested contaminant RNAs can still be captured and erroneously identified as resident transcripts. Here we introduce Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq) as a new subcellular transcriptomics approach that efficiently bypasses this caveat. CoLoC-seq leverages classical enzymatic kinetics and tracks the depletion dynamics of transcripts in a gradient of an exogenously added RNase, with or without organellar membranes. By means of straightforward mathematical modelling, CoLoC-seq infers the localisation topology of RNAs and robustly distinguishes between genuinely resident, luminal transcripts and merely abundant surface-attached contaminants. Our generic approach performed well on human mitochondria and is in principle applicable to other membrane-bounded organelles, including plastids, compartments of the vacuolar system, extracellular vesicles, and viral particles.

Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) Probes the Global Localisation Topology of Organelle Transcriptomes

“…Note: Although the fragmentation step may be expected to destroy the strand-specificity of the protocol, our mapping results ( Jeandard et al, 2023 ) showed that the first 5′-adapter ligation (step G2) largely determines the strandedness of the reads, which permits unambiguous transcript assignment and quantification. Other strategies to enforce the preservation of the strand information can also be implemented (J.…”

“…We evaluated the feasibility and performance of CoLoC-seq on the well-studied mitochondrial transcriptome of human embryonic kidney 293 (HEK293) cells ( Jeandard et al, 2023 ). To this end, two biological replicate series of CoLoC-seq and Mock CoLoC-seq samples were analysed by northern blotting and RNA-seq.…”

“…Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), which marries the accessibility and generality of fractionation-based approaches with the selectivity and robustness of proximity labelling techniques ( Jeandard et al, 2023 ). In a standard CoLoC-seq pipeline ( Figure 1 , the blue branch), a preparation of intact organelles of interest is split into a suite of samples subjected to a gradient of RNase concentrations.…”