1The Weizmann Institute of Science, Rehovot, Israel; 2The Hebrew University of Jerusalem, Jerusalem, Israel Processing of pre-mRNA in the nucleus occurs while the transcript is packaged with spliceosomal U snRNPs and protein splicing factors in a large supraspliceosome complex, which has overall dimensions of 50x50x35 nm and a mass of 21 mDa. By a combination of molecular and electron microscopy (EM) techniques we have now demonstrated that the complex is composed of four similar spliceosomal subunits and an additional substructure, which are associated with a single pre-mRNA molecule that connects the substructures. Structural analysis of the supraspliceosome in its native aqueous state by cryo-EM revealed holes within the spliceosomal subcomplexes, whose role is yet unclear. Cryo-EM also revealed fibrils formed by the pre-mRNA, which interconnect the subunits. The additional fifth substructure is attributed to additional pre-mRNA processing activities, since we have shown that 5’- and 3’-end components, as well as A-to-I editing enzymes, are associated with the supra-spliceosome. The supraspliceosome can thus be viewed as the native pre-mRNA processing machine. The supraspliceosome is a molecular machine required to process the pre-mRNA in an accurate, and regulated manner, yielding functional mRNA. It provides a frame onto which the pre-mRNA is folded, allowing juxtaposing exons about to be spliced, while introns are looped out of each of the respective spliceosomes. It enables “communication” between the spliceosomes and coordinated splicing of a multiintronic pre-mRNA. It can also account for regulated alternative splicing, which is a major source of protein versatility in mammals.
