Research
Molecular mechanism of pre-mRNA splicing
We study the molecular mechanism of the pre-mRNA splicing reaction using the yeast Saccharomyces cerevisiae as a model system. We have identified two protein complexes, NTC and NTR, and several other protein factors involved in various steps of spliceosome assembly. NTC contains at least eight protein components, and is required for spliceosome activation. NTR contains two protein factors in addition to the DExH-box RNA helicase Prp43, and is involved in spliceosome disassembly after completion of the splicing reaction.
Activation of the spliceosome involves sophisticated structural rearrangement of the spliceosome including the release of U1 and U4, and new base-pair formation between U6 and U2, and between U6 and the 5¡¦ splice site. UV-cross-linking analysis reveals that U5 and U6 interact with pre-mRNA in a dynamic manner, spanning over a large region of U6 and the 5¡¦-exon sequence, in the pre-activated spliceosome. Upon dissociation of U4, NTC is required for defining specific interactions between U6 and the 5¡¦ splice site and between U5 and the 5¡¦-exon sequence to stabilize the catalytic structure of the spliceosome. The function of individual NTC components in mediating such structural change is under investigation.
Disassembly of the spliceosome after completion of the catalytic reaction is necessary for recycling of splicing factors to facilitate efficient splicing. The DExH-box RNA helicase Prp22 has been shown required for the release of the mature mRNA, and Prp43 required for the release of lariat-intron as the final step of the process. We have found that Prp43 is recruited to the spliceosome by U5 through a series of protein-interactions, linking U5 component Brr2-Ntr2-Ntr1-Prp43. The mechanism underlies the disassembly process and how NTR is prevented into pre-mature spliceosome are being investigated.
