Structural studies of the endogenous spliceosome - The supraspliceosome.

Pre-mRNA splicing is executed in mammalian cell nuclei within a huge (21MDa) and highly dynamic molecular machine - the supraspliceosome - that individually package pre-mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature. Detailed structural analysis of this huge and complex structure requires a stepwise approach using hybrid methods. Structural studies of the supraspliceosome by room temperature electron tomography, cryo-electron tomography, and scanning transmission electron microscope mass measurements revealed that it is composed of four native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA. It also elucidated the arrangement of the native spliceosomes within the intact supraspliceosome. Native spliceosomes and supraspliceosomes contain all five spliceosomal U snRNPs together with other splicing factors, and are active in splicing. The structure of the native spliceosome, at a resolution of 20Å, was determined by cryo-electron microscopy, and a unique spatial arrangement of the spliceosomal U snRNPs within the native spliceosome emerged from in silico studies. The supraspliceosome also harbor components for all pre-mRNA processing activities. Thus the supraspliceosome - the endogenous spliceosome - is a stand-alone complete macromolecular machine capable of performing splicing, alternative splicing, and encompass all nuclear pre-mRNA processing activities that the pre-mRNA has to undergo before it can exit from the nucleus to the cytoplasm to encode for protein. Further high-resolution cryo-electron microscopy studies of the endogenous spliceosome are required to decipher the regulation of alternative splicing, and elucidate the network of processing activities within it.