What happens to U4 during the C complex formation?

During the formation of the C complex in the spliceosome assembly, U4 is released from the complex. The C complex is the step in the spliceosome where the spliceosomal components have assembled and are ready for the catalytic activity of splicing to occur. Prior to C complex formation, U4, along with U6, forms a heterodimer and is initially part of the spliceosomal machinery.

As the complex transitions to the C stage, a critical rearrangement happens where U4 is released in order for U6 to interact with U2 and facilitate the activation of the spliceosome for the splicing reaction. This release is necessary because U4's presence would inhibit the proper function and catalytic activity required for splicing. Thus, it is the release of U4 that is a defining feature of the C complex formation, allowing the spliceosome to proceed with its function in pre-mRNA splicing.