Periodic Reporting for period 1 - SplicEcomplex (Deciphering the structure and dynamics of the early spliceosome assembly)
Reporting period: 2018-04-01 to 2020-03-31
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.
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.
My work has focused on the study of the bridging factor FBP11 which connects the 5´ and 3´splicing sites in the early spliceosomal arrangement. FBP11 is a multimodular protein that contains several WW and FF domains that serve as binding platforms for many different proteins. We have characterized the structure and dynamics of most of the domains and moreover we have studied their arrangement in a larger context by the combination of different structural techniques in solution (Nuclear Magnetic Resonance spectroscopy, Small Angle X-Ray Scattering) and by X-ray crystallography. This integrative structural approach shed light to the atypical domain distribution of FBP11. At the same time, we investigated how FBP11 interacts with the different factors that play an important role in early spliceosomal assembly. We have narrowed the interaction of WW domains of FBP11 to a short peptide of SF1 and analysed its affinity and selectivity. In addition, we have determined the binding sites of FBP11 FF domains to Luc7 and 70K proteins. Finally, in order to obtain high resolution structural information of the E complex we have performed the assembly of different components and studied the complexes by cryo electron microscopy.
Initial results of this work have been published. Key findings will be published in the near future and have been disseminated in several international conferences and meetings, reaching to a high number of researchers. Results obtained have also been disseminated in several outreach activities highlighting the importance of basic research to the non-academic audience in Europe.
Initial results of this work have been published. Key findings will be published in the near future and have been disseminated in several international conferences and meetings, reaching to a high number of researchers. Results obtained have also been disseminated in several outreach activities highlighting the importance of basic research to the non-academic audience in Europe.
The impact of the research performed during the action on the scientific field, consisting on the advances made to determine the early complex arrangement and the role of the bridge factor FBP11 has on it, adds to a profound understanding of early spliceosomal assembly which is in end essential to understand the regulation of a fundamental step in biology as is the splicing of mRNA. As it is known, misregulation of the correct splicing leads to different alternative splicing outcomes, which is implicated in severe genetic diseases and cancer. Thus, all efforts to understand this important mechanism have a huge impact on society. Our work represents an important contribution to expand the state-of-the-art knowledge on spliceosomal assembly, which will aid other research groups, more focussed on medical and health applications, in the development of better diagnostics, more effective therapeutic approaches and in finding new ways to prevent these diseases.