Biological processes, such as cellular growth and differentiation, are controlled by the expression of genetic material within a cell. Accumulation of alterations in the expression of a number of critical genes is the mechanism by which cells become cancerous. Characterizing the molecular details of gene expression in both normal and aberrant situations is an important part of dissecting this mechanism.

The Essential Role of RNA Splicing

Dynamics of the Spliceosome

EM of C complex. 2D images (little blobs) to 3D structure (colored)

In eukaryotes, RNA splicing is an essential step in gene expression. This involves the excision of non-coding intervening sequences, called introns, and the ligation of functional coding regions, called exons. The precise removal of introns is required to create continuous coding sequences with the correct genetic information for subsequent protein translation. In addition to this basic function, splicing provides a key point of regulation for gene expression by means of the differential inclusion of exons - a process called alternative splicing. Alternative splicing allows a single gene to produce a set of distinct but related proteins. This flexibility significantly increases the size and complexity of the proteome.

Dissecting the Spliceosome

The cellular machine responsible for RNA splicing is the called spliceosome. This multi-megadalton macromolecular complex rivals RNA polymerase II holoenzyme and the ribosome in size, complexity and importance for gene expression. It is composed of five small ribonucleoprotein (snRNP) particles and a large number of additional protein factors that assemble on nacscent transcripts in the nucleus to perform the splicing reaction. The snRNPs (known as U1, U2, U4, U5 and U6) are also multi-component complexes, containing one small stable RNA molecule (snRNA) and five or more individual proteins. Jurica and her laboratory are using the tools of structural biology to characterize the architecture of the entire spliceosome, as well as to provide detailed information concerning its approximately 200 components.

The large size and complexity of many macromolecules result in several challenges for structural determination. With the spliceosome, the task is even more formidable due to its dynamic assembly, lack of symmetry and possible structural flexibility. It is, therefore, necessary to combine various structural techniques in order to create an architectural representation of this complicated machine. Jurica and her colleagues use cryo-electron microscopy for ultrastructure studies of large splicing complexes and X-ray crystallography to determine high-resolution structures of individual components. Results from these endeavors are combined with computational modeling and experimentally derived restraints from biochemical studies of spliceosomes to build a three-dimensional model that will further our understanding of the mechanistic underpinnings of pre-mRNA splicing. This multi-faceted approach can also be applied to other macromolecular complexes.