Background
Transcripts from most mammalian genes are synthesized as precursor mRNAs (pre-mRNAs), from which non-coding introns must be spliced out in the nucleus to produce mRNAs with continuous protein-coding information that can be exported to the cytoplasm and translated into protein. Through alternative splicing, introns expand proteomic diversity by allowing a single gene to encode multiple protein isoforms with distinct activities.
pre-mRNA splicing was discovered in 1977 by electron microscopy and is performed by the spliceosome - a dynamic assembly of RNA and proteins. The spliceosome performs two sequential transesterifications to effect pre-mRNA splicing and undergoes complex rearrangements between these two steps. Despite its crucial role in gene expression, the molecular structure of the spliceosome had remained elusive for decades. In recent years, in the lab of the late Kiyoshi Nagai, Sebastian Fica, Wojtek Galej, and Max Wilkinson took advantage of the "resolution revolution" and used electron cryo-microscopy to solve structures of key intermediates of the yeast and human spliceosome during the catalytic stage. These structures, together with those solved by the groups of Yigong Shi and Reinhard Luhrmann, revealed the conformation of the RNA-based active site and the molecular rearrangements between the two catalytic steps, showing how splice sites are recognised through conserved RNA-RNA pairing in both yeast and mammals.
Many human-specific proteins modulate the conformation of the spliceosome during catalysis and influence how a single gene produces multiple mRNAs through alternative splicing. Despite many of these human proteins being implicated in an array of pathologies, including cancer, their specific roles and mechanisms of action remain largely obscure. We have recently discovered that several of these proteins bind the human spliceosome during the catalytic stage and promote mRNA synthesis.
Goals
We aim to dissect in vitro the catalytic stage of human pre-mRNA splicing and combine biochemistry and electron cryo-microscopy to understand the mechanisms that govern spliceosome dynamics, splice site selection, and proofreading during the catalytic stage. We seek to determine how human-specific splicing factors regulate spliceosome dynamics and splice site choice during catalysis. We are particularly excited by the possibility that human spliceosomes employ transcript-specific factors during the catalytic stage and we are interested in understanding whether such factors may regulate alternative splicing during exon ligation. To understand spliceosome dynamics, we seek to determine the role of the several human-specific DEAH-box ATPases that associate with the catalytic spliceosome, and which may regulate spliceosome rearrnagements and mRNA synthesis in a transcript-specific manner.
To extend our in vitro findings, we are also collaborating with Jernej Ule's lab at The Francis Crick Institute to perform protein-RNA crosslinking experiments to understand the role of these factors genome-wide in vivo in cell culture. More broadly, we seek to purify native spliceosomes stalled at various stages of assembly and catalysis from different tissue types for mass spectrometry, protein-RNA crosslinking, and structural studies, with the goal of delineating the mechanisms through which splicing factors that act during the catalytic stage regulate splicing in a tissue-specific manner. We hope that these studies would also lead to the discovery of novel splicing factors and of potential therapeutic targets for modulating pathological splicing patterns.
