Investigation of Protein-protein Interactions within the Human Spliceosomal U4/U6.U5 tri-snRNP Particle

Abstract

Two-hybrid data allowed me to define the regions through which proteins 220K, 200K, and 116K interact with each other. These regions include the N terminal and C-terminal domains of protein 220K, the second helicase domain of 200K, and the C-terminal domain of 116K. Strikingly, most of the binding domains are also identified in the interactions of the yeast orthologue proteins Prp8p, Brr2p, and Snu114p.Protein 102K interacts with several particle-specific proteins of both the U5 and the U4/U6 snRNP, and thus very probably acts as a bridging or scaffolding protein in the formation or structural stabilisation of the tri-snRNP. The interactions with U5-220K, 200K and 116K allow the 102K protein to bind stably to the U5-snRNP particle. The 102K protein is the only U5-specific protein identified in this work that interacts with U4/U6-specific proteins 61K and 90K. The former interaction has been proved to be critical for the formation of the tri-snRNP. One of the missense mutations (A194E) in the 61K protein responsible for the disorder retinitis pigmentosa has been shown a reduced interaction with the 102K in this study. The possible mechanisms of this are discussed. Mutational analysis showed that the TPR repeats of 102K protein are each dedicated to the interactions with specific partners. All repeats participate in the interaction with the U4/U6-61K, whereas only the first nine repeats interact with 110K, 200-4 fragment of 200K, and the 220-1 fragment of 220K.The U4/U6-90K protein interacts with 60K within the 20K 60K 90K heterotrimer and contacts the U6 snRNA in the stem II region in the U4/U6-snRNP particle. In this study, it was shown that this protein interacts with human recycling factor U6-p110 and thus functions in the recycling of U4/U6 snRNPs. Mutational analysis showed that the C-terminal domain of 90K, comprising amino acids 417 683, is responsible for this binding. During the formation of tri-snRNP, the U5-102K protein interacts with 90K protein in the same domain, suggesting that U5-102K might function in the release of U6-p110. Protein 90K also interacts with U2-associated protein SPF30/SMNrp through its N-terminal region, and therefore functions in the recruitment of tri-snRNP into pre-spliceosome.The tri-snRNP-specific 110K protein interacts with U4/U6-90K, U5-102K and U5 200K through the C-terminal region lacking an RS domain. Since the absence of 110K protein does not compromise the stability of the tri-snRNP, it appears reasonable that anchoring 110K to these proteins may be required to properly position 110K for its contribution in connecting the tri-snRNP to the pre-spliceosome.The U5-52K protein interacts with the U5-102K and 15K proteins, suggesting that these interactions contribute to its integration into the U5 particle. Binding studies performed with 52K deletion mutants revealed that the N-terminal two-thirds of 52K interact with the 102K protein, whereas its C-terminal GYF-domain binds the 15K protein. The GYF domain has been characterised previously as a polyproline-targeting molecule. As the 15K protein lacks a proline-rich tract, these data indicate for the first time that a GYF-domain can also engage in specific protein-protein interactions in a polyproline-independent manner. The crystallography study of the 52K GYF domain in complex with 15K, a cooperative work with the laboratory of Prof. Dr. R. Ficner at the University of Göttingen, showed that the 15K protein makes contact with the distinctive surface of GYF domain, as does the proline-rich target. The data from this study and others demonstrated that the 52K protein is the only 20S U5-specific protein that is not integrated into the tri-snRNP.On the basis of the data obtained in this work, I propose a model of the assembly of the U4/U6.U5 tri-snRNP.