A collaborative study between Prof. Torben Heick Jensen’s and Dr. Sander Granneman’s laboratories at Aarhus University and Edinburgh University, respectively, now reveals new insights into the spatiotemporal dynamics of co-transcriptional RNA-protein interaction.

The gold standard for studying such interactions in vivo is so-called UV-mediated ‘cross-linking and immunoprecipitation (CLIP)’ technology, which essentially involves irradiating cultured cells with UV light to permanently cross-link proteins to the RNAs they are interacting with. Subsequently, cognate RNAs can then be identified by purifying the ribonucleoprotein (RNP) complexes and performing high-throughput RNA sequencing. However, this technique only captures the steady-state protein-RNA binding profiles of the cultured cells, preventing dynamic analysis of protein-RNA interactions.

Developing a new technique - tiCLIP

To meet this challenge, Cordiner et al., developed a technique coined ‘temporal iCLIP (tiCLIP)’, which monitors protein-RNA interactions in a narrowly time-resolved window after the re-initiation of transcription, proceeding its prior shut-down. tiCLIP was then applied to the RNA-export factor ALYREF, the RNA-decay factor RBM7, and the cap-binding complex components CBP20 and CBP80 (see figure).

The study found that, regardless of their roles in RNA fate, all tested proteins interact with the nascent RNA as it exits the transcription machinery. These early interactions with nascent RNA may be an initial phase of transient RBP searching for higher affinity anchoring sites.

Additionally, the results demonstrate that the process of pre-mRNA splicing temporally separates the association of RNA-export and -decay factors binding to splicing intermediates.

Finally, the spatiotemporal resolution achieved by tiCLIP revealed underappreciated processing intermediates of small nucleolar RNA (snoRNAs) bound by the RNA decay factor, RBM7, which might shed light on the mechanism by which mature snoRNAs are processed.

The researchers are now working to further optimize the tiCLIP technology and to apply it to additional RBPs. One goal is to reach a resolution where CLIP can be used on tissue samples.