Dissection of U4 snRNA functional domains using in vitro reconstitution.

Splicing, the removal of non-protein-coding introns from pre-mRNA, is a critical step in eukaryotic gene expression, yet this process is still far from being fully understood. Investigation of the splicing mechanism has been hindered by its complexity. The spliceosome, the dynamic ribonucleoprotein complex which catalyzes splicing, contains over 100 molecular components, some of which are organized into small nuclear ribonucleoprotein particles (snRNPs). Each snRNP is composed of a small nuclear RNA (snRNA U1, U2, U4, U5, or U6) and its associated proteins. While the general roles of U1, U2, U5, and U6 are reasonably well understood, the function of U4, though essential for splicing, remains much less clear. It has been demonstrated that U4 forms an extensive base-pairing interaction with U6, but the function and mechanism of formation of this interaction have not been established. One reasonable hypothesis is that interaction of U4 and U6 allows U4 to activate U6 for assembly onto the pre-mRNA. Furthermore, there is only a limited knowledge of the proteins associated with U4, where they bind, and what functions they may perform during snRNP and spliceosome assembly and splicing catalysis. Determination of the structure of the U4 snRNP and its role is splicing has been hindered by lack of an in vitro system allowing U4 reconstitution analyses. To facilitate molecular dissection of the structure and function of U4 in splicing, I have developed an in vitro assay for reconstitution of functional U4 snRNPs. Endogenous U4 was depleted from yeast splicing extract through oligonucleotide-directed RNase H degradation, and optimal conditions for this reaction were identified. The most important factor for efficient U4 degradation was to perform the reaction under conditions of active pre-mRNA splicing, as the splicing process was found to increase accessibility of the targeted region of U4. Depletion of U4 resulted in an early block in spliceosome assembly, strongly inhibiting splicing activity. Subsequent addition of