The central dogma in molecular biology, postulated by Francis Crick in 1958, describes the sequential and unidirectional flux of information from DNA to proteins. Presumed at the beginning as being a mere messenger, the intermediate RNA was shown later to be a key factor in protein synthesis variability and regulation. Currently, our knowledge of mRNA metabolism is more expanded; precursor (pre-) mRNA undergoes numerous distinct co/post-transcriptional modifications in eukaryotes, in which the mRNA, once it has been correctly processed, has to travel from the nucleus to the cytoplasm. Among the modifications that occur in the eukaryotic nucleus, pre-mRNA splicing appears as one of the most intriguing and complex aspect of RNA processing. The pre-mRNA is edited for removal of noncoding sequences (introns) and ligation of the coding sequences (exons), giving rise to an RNA sequence that encodes the corresponding protein sequence. In addition, many pre-mRNAs can be spliced in different ways, generating a great variability of processed mRNAs (alternative splicing); a prevalent process in eukaryotes that expands their complexity by increasing the number of proteins that can be encoded in a single gene. Disruption of normal splicing patterns is linked to several human diseases, ranging from metabolic syndromes, muscular dystrophies, neurodegenerative disorders, cardiovascular diseases and tumor development. Thus, a better understanding of the molecular mechanisms that perform and regulate splicing process is key to find new ways to prevent these diseases and develop better diagnostics and more effective therapeutic approaches (EU Horizon 2020 Strategy in Health).
Splicing of pre-mRNA is performed by a large ribonucleoprotein (RNP) complex, the spliceosome, and consists on a precise and coordinated process in which many different components undergo a very dynamic assembly / remodelling / dissociation cycle in which the pre-mRNA recruits the different snRNPs at conserved intron sequences, at different times and in a precise sequence in order to correctly perform the two catalytic steps of splicing. A critical early aspect that directs splicing is the correct spatial disposition of the 5’ and 3’ splice sites in the intron sequence prior to splicing, which is defined as an early step in the cycle with the assembly of spliceosome components in 5' (U1snRNP) and in 3' (SF1-U2AF heterodimer) and their physical connection through bridge proteins (complex E).
Despite of previous structural studies on isolated E complex components and all the exciting and novel cryo-EM structures of several spliceosome catalytic steps that have been obtained in the past few years, there is still lack of information on the whole E complex structural arrangement. Understanding molecular details of E complex and its assembly is essential to reveal mechanisms underlying the regulation of alternative splicing, where in most cases regulatory RBPs, modulate spliceosome assembly at these early steps. Thus, the main goal of this proposal is to obtain high-resolution information on the structure and dynamics of the cross-intron arrangement spliceosome in the E complex stage.
