Abstract
Spinal Muscular Atrophy (SMA) is a neurodegenerative disease that is caused by mutations in the ?survival of motor neurons? gene (SMN). Initial experiments showed that in vivo the SMN protein (SMN) is associated with components of spliceosomal U snRNPs. U snRNPs are small RNA-protein complexes that form functional subunits of the spliceosome which is the cellular machinery responsible for processing nuclear pre-mRNA into mature mRNA.
The snRNPs U1, U2, U4 and U5 contain a set of seven Sm-Proteins (SmB/B?, D1, D2, D3, E, F and G) that associate at the single stranded uridyl-rich Sm-site of the snRNA thereby forming the Sm-core domain. This domain builds the structural framework that is common to the different U snRNPs. Experimental evidence suggested that the formation of the Sm-core in vivo (in contrast to existing in vitro data) is dependent on factors such as SMN that are not found in the mature particles.
The putative function of SMN during U snRNP assembly was investigated using biochemical approaches. It was shown that SMN interacts directly with spliceosomal Sm-Proteins via its central Tudor-domain. Thus, the Tudor-domain, a sequence motif of so far unknown function mediates protein-protein interactions. Injection experiments in Xenopus laevis oocytes revealed that antibodies that specifically block the interaction between the Tudor-domain of SMN and Sm-Proteins inhibit the formation of the Sm-core domain in vivo. Therfore SMN is an essential factor for the assembly of U snRNPs in vivo. Furthermore, the analysis of a SMA causing mutation within the Tudor-domain revealed reduced interaction between SMN and Sm-Proteins as a biochemical defect possibly occuring in SMA patients.
Fractionation of cell extracts showed that SMN in vivo is incorporated into macromolecular complexes that are likely to mediate the function of SMN during U snRNP biogenesis. These complexes share a common structure of eight proteins which is formed by SMN, the known factors SIP1, U1A and Gemin3/dp103 and the four newly identified SMN associated proteins GIP1/Gemin4, unrip, Hsc 70 and p175. By establishing a cell free system that allows for the SMN dependent reconstitution of U snRNPs it was shown that U snRNP assembly in vivo (contrary to existing in vitro data) is an energy dependent process.
With this system at hand and the availability of SMN-complexes in preparative amounts it will be possible to characterize the mechanistic aspects of U snRNP assembly in vivo and to analyze the functional contributions of the different SMN-complexes and their components. |
