Structural basis of branching during RNA splicing.

Branching is a critical step in RNA splicing that is essential for 5' splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine at the branch point. However, there are no structures of any splicing complex with the adenosine nucleophile docked in the active site and positioned to attack the 5' splice site. Thus we lack a mechanistic understanding of adenosine selection and splice site recognition during RNA splicing. Here we present a cryo-electron microscopy structure of a group II intron that reveals that active site dynamics are coupled to the formation of a base triple within the branch-site helix that positions the 2'-OH of the adenosine for nucleophilic attack on the 5' scissile phosphate. This structure, complemented with biochemistry and comparative analyses to splicing complexes, supports a base triple model of adenosine recognition for branching within group II introns and the evolutionarily related spliceosome.

Scaffold-enabled high-resolution cryo-EM structure determination of RNA.

Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA, which we demonstrate with the 86-nucleotide thiamine pyrophosphate (TPP) riboswitch, and visualizing the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch undergoes a large-scale conformational change upon ligand binding, illustrating how small molecule binding to an RNA can induce large effects on gene expression. This study both sets a new standard for cryo-EM riboswitch visualization and offers a versatile strategy applicable to a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.