Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 2005

Functional Proteomics and Structural Dynamics of Mammalian Spliceosomes

The U2-dependent (major) spliceosome is a large molecular machine that consists of the snRNPs U1, U2, U4/U6, and U5 and more than 150 non-snRNP splicing factors, and undergoes multiple structural rearrangements during pre-mRNA splicing. To learn more about changes in the spliceosome?s protein composition and RNP remodelling events during the spliceosomal cycle, we have immunoaffinity purified spliceosomal complexes at defined stages of assembly or function (e.g. complexes A, a pre-catalytic B complex and the catalytically activated B* spliceosome) and have characterized their protein compositions by mass-spectrometric methods. These studies revealed significant changes in protein composition during the transition from one spliceosomal complex to another. In addition, they provided evidence that the U5 snRNP is restructured during catalytic activation of the spliceosome and is released from the post-spliceosomal complex as a 35S U5 RNP particle. We are currently using RNAi interference to investigate the function of newly identified spliceosomal proteins in pre-mRNA splicing in HeLa cells.

The ultrastructure of purified native B complexes have been studied by single-particle electron cryomicroscopy; complexes were mainly triangular and omega shaped with a diameter of ∼370Å.

We have also determined the 3D structure of the human U11/U12 di-snRNP by cryo-EM. The U11/U12 di-snRNP is a major component of the U12-dependent (minor) spliceosome and interacts simultaneously with the 5?splice site and the branch point region of a U12-dependent intron. Thus, it forms a molecular bridge that functionally pairs intronic sequences that are important for the first step of splicing. SF3b, a heptameric protein complex of the U11/U12 di-snRNP, is expected to play a central role in recruiting the di-snRNP to the branchpoint region similar to its role in the major spliceosome in which most of the SF3b proteins crosslink to pre-mRNA including SF3b49, SF3b155 and p14. We have recently shown by electron cryomicroscopy (cryo-EM) that isolated SF3b resembles a bivalve shell with p14 located in the interior of the complex. This remarkable architecture raised the question of how p14 might interact with the intronic branchpoint (Science 300, 980-984 (2003)).

The 3D reconstruction of native U11/U12 di-snRNP revealed a size of 26 × 15 × 17 nm for this particle. We directly localized the HEAT repeats of SF3b155 and the RNA recognition motifs of SF3b49 and p14 based on their known structural elements as well as the N-terminus of SF3b155 by immunolabeling. According to this data, both shell halves of SF3b were located in the outer wall of the U11/U12 di-snRNP. Significantly, the conformation of SF3b bound to the U11/U12 di-snRNP differs from that of isolated SF3b. The two shell halves of SF3b adopt a closed form in isolated SF3b, whereas SF3b rearranges into a more open conformation upon integration into the di-snRNP. Based on this data a putative model of the pre-mRNA binding to the U11/U12 di-snRNP is proposed.

Reinhard Luhrmann*
Henning Urlaub
Holger Stark

Max-Planck-Institute for Biophysical Chemistry
Dept. of Cellular Biochemistry
Am Fassberg 11,Göttingen, Germany

*Phone: 0049-551-201 1407
Fax 0049-551-201 1197
Email: reinhard.luehrmann@mpi-bpc.mpg.de