Cryo-EM structural analysis of spliceosome biogenesis

The spliceosome is a multi-megadalton enzyme that catalyses excision of non-coding introns from pre-mRNA. Spliceosomes are assembled from five small nuclear ribonucleoprotein particles (snRNPs), which contain unique small nuclear RNA (snRNA) and are bound by the heptameric Sm-ring and from additional non-snRNP factors. In humans, snRNP biogenesis requires ±15 proteins, including the 9-subunit survival of motor neuron (SMN) complex, which carries out the regulated and specific loading of the Sm ring onto snRNA. Although the Sm-ring assembly was connected to several diseases, the complete structures of either SMN complex alone or interacting with Sm-ring and the snRNP as a whole are lacking, leaving many questions on snRNP assembly unsolved.

We reconstituted key steps in snRNP biogenesis in humans and determined their structures in sub-atomic resolution using cryo-electron microscopy. We used Cryo-EM to analyze the loading steps of the Sm-ring onto a model snRNA with the help of the SMN complex and identified interacting partners while assigning new functions to them. Furthermore, we visualized the structural transitions within the assembled snRNP that were supported by structure-guided biochemical and tissue-culture validations of SMN complex function. This work has provided us with exciting insights into the molecular events and choreography of snRNP assembly.