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In addition to these methods to profile translation in single cells, there are also several single-cell technologies to measure mRNA features that regulate translation. There wasn’t space to go into detail, but we also included a brief overview of these methods

Translation ultimately revolves around an mRNA template. Determining the amounts and states of these templates and how they interact with regulatory machinery is important to understand translational regulation

Translation machinery is spatially distributed to modulate expression patterns. RIBOmap from Xiao Wang’s group measures the spatial localization of mRNAs being translated in tissue slices. Signal is only generated when the rRNA-padlock splint, padlock, and RCA primer are colocalized

Ribo-STAMP from Gene Yeo’s group instead records ribosome association with RNA edits. Here, a RPS2-APOBEC1 fusion is transiently expressed that is localized to mRNAs by translating ribosomes. Highly translated transcripts will thus accumulate C-U mutations

T&T-seq by Kehkooi Kee’s group splits the lysate from a single oocyte, with part used for scRNA-seq, and the other part is used in a RiboLace pull down to select transcripts being actively translated. Highly translated transcripts will be more abundant in the pull down

Ribosome profiling isn’t the only way to measure ribosome-transcript interactions though!
An alternative approach is to quantify changes in the degree to which ribosomes are associated with transcripts

These techniques are still in their infancy, but they’ve already made it possible to interrogate translation as a highly dynamic layer of gene regulation in previously inaccessible systems

These new technologies allowed translation and its regulation to be studied in dynamic systems such as dividing cells, limited samples from early embryos, and rare primary cells.

Modifications to this workflow by us (scRibo-seq), the Cenik lab (Ribo-ITP), and Wei Xie (Ribo-lite) increased the sensitivity of traditional ribosome profiling by using different footprint enrichment strategies and efficient library preparation chemistries

Ribosome profiling provides a high-resolution snapshot of active translation. By arresting ribosomes on transcripts and digesting away unprotected RNA, you create short ribosome-protected footprints. Sequencing these footprints gives the numbers and locations of ribosomes