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Mechanisms of alternative pre-mRNA splicing regulation in cancer and pluripotent cells

Periodic Reporting for period 3 - MASCP (Mechanisms of alternative pre-mRNA splicing regulation in cancer and pluripotent cells)

Reporting period: 2018-10-01 to 2020-03-31

Alternative splicing of messenger RNA precursors is a mechanism of gene regulation that expands the coding capacity and regulatory opportunities of genomes, contributes to cell differentiation and pluripotency and its deregulation promotes cancer progression, as evidenced by the frequent occurrence of cancer-associated mutations in splicing factors, which are also targets of anti-tumor drugs. Despite its prevalence and relevance, the underlying mechanisms of regulation remain poorly understood. This proposal aims to develop and apply systematic approaches that will unweave the complex network of functional interactions that underlie alternative splicing and its relevance for the control of cell proliferation and/or pluripotency in cancer and induced pluripotent stem (iPS) cells. Progress in this area can contribute to reveal the molecular logic governing a key layer of gene regulation and has the potential to discover novel factors and regulatory circuits that trigger or modulate cell growth, differentiation and cancer progression.

- Why to do this project? While we can routinely sequence genomes, we still cannot predict the alternatively spliced products of gene expression, neither during normal development nor in disease. We ignore even the basic rules of splice site selection, to which the cell dedicates one of its most elaborate molecular machineries. Therefore understanding the process of alternative splice site selection and its rules remains a challenge of fundamental and biomedical importance.
- Why to do it now? Because recent technical developments, including contributions from our group, provide unprecedented opportunities to unweave the network of functional interactions within the spliceosome, and of the splicing machinery with other cellular processes, and to investigate mechanisms of alternative regulation through the equivalent of high-throughput genetic analysis in mammalian cells. Thus, the proposal aims to carry out frontier research through ground-breaking, ambitious and interdisciplinary efforts aligned with the ERC goals.
- Why can it change the field? Combination of network analysis with transcriptome-wide sequencing will provide a rich, open resource of information that researchers will be able to use to explore mechanisms of alternative splicing regulation. High-throughput assessment of sequence variants will also have a significant impact, including the evaluation of the effects on splicing of disease-associated mutations.
- Why can it have an impact beyond its field? Because concepts, technologies and reagents generated during the project will be applicable to other processes of gene expression, from transcriptional/epigenetic control to translation regulation, in a variety of biological systems. Furthermore, the results of the project can significantly advance our knowledge of novel regulatory circuits involved in cell proliferation and differentiation, cancer and cellular reprogramming. The emergence of therapeutic approaches –currently in clinical trials for the treatment of Spinal Muscular Atrophy and some cancers- based upon modulation of splicing by modified antisense oligonucleotides or small compounds are good reminders that deep understanding of mechanisms of splicing regulation has a bright future ahead.
The bulk of the actions carried out in the first 18 months of the MASCP project can be summarized as follows:
a) Procurement of materials and RNA-seq analysis for building transcriptome-wide splicing regulatory networks. This effort represents the core of the MASPC project, as it serves as the basis for reconstructing maps of functional interactions between components of the spliceosome. RNAs corresponding to the knock down of each individual component of the spliceosome (both Major and Minor spliceosomes), splicing regulatory factors, RNA modifying enzymes and chromatin remodelling factors influencing RNA processing in a cancer cell line (HeLa, 304 knock downs in total) were isolated and subject to paired-ended RNA sequencing. Sequencing (100 M reads/sample) was completed in April 2017 and datasets were organized, merged, aligned and, after quality-control, alternative splicing was quantified using novel computational pipelines (VAST-tools/SanJuan). Special efforts were made to optimize the datasets to be able to later on integrate RNA-seq data from a collection of knock downs corresponding to every protein-coding gene in the human genome, thus facilitating the investigation of links between splicing regulation and virtually any other cellular pathway. In parallel, work was carried out to establish a similar knock down / RNA analysis system for two human iPS cells (KYOU and KIPPS). While a significant number of technical hurdles needed to be addressed, our datasets represent the most comprehensive collection of RNA-seq data available to systematically monitor the function of each individual component of the splicing machinery and as such they can provide a unique resource to probe the function of the spliceosome.

b) Detailed analysis of regulatory mechanisms, including the establishment of methods for high-throughput analysis of regulatory sequences and factors. In parallel with the systematic sequencing efforts above, mechanistic studies were carried out to understand the function of sequences and regulatory factors identified in our previous functional network studies, including:

b.1. Regulation of splice site recognition. We have developed methods to systematically assess the effects of nucleotide substitutions in alternatively spliced exons and applied this for the evaluation of alternative splicing of the apoptotic receptor Fas (Julien et al, Nature Comm 2016) as well as NUMB exon 9 (relevant for cancer cell proliferation) and SMN1 / SMN2 (relevant for Spinal Muscular Atrophy). The results reveal high dense regulatory content of alternative exons and extensive epistatic interactions in exon definition. Work on particular subnetworks involved in 3' splice site recognition led to the characterization of a complex essential for cell cycle progression (Martin et al, in final phases of preparation) and another complex of proteins interacting with the factor ZRSR1 that plays a role in mouse spermatogenesis (Horiuchi et al, submitted to Cell Reports). Links between transcription / R loop formation and alternative splice site selection have also been detected and the underlying mechanisms are currently under investigation.

b.2. Modulation of splicing by core components of the splicing machinery. Structure/function analyses revealed that the splicing regulatory factor RBM5 contacts the core splicing factor SmB/B'/N to modulate alternative splicing, revealing the potential of core components of the Sm complex for influencing splice site selection (Mourao, Bonnal et al, eLife 2016). Expression of mutants in core splicing factors associated with Retinitis Pigmentosa revealed splicing alterations in other genes mutated in the disease, offering a possible molecular link between the mutations in splicing factors and relevant target genes (Rogalska et al, in preparation). Work on small molecules targeting the key core splicing factor SF3B1 provided compounds of improved stability and activity (Makowski et al, ACS Chem Biol 2016), as well as dissected 3' splice site sequences that modulate drug response, revealing also significantly different splicing regulation effects by structurally different drugs (Vigevani et al, submitted to Nat Chem Biol).

b.3. Integration of alternative splicing with cellular programs of cell proliferation and pluripotency. Work in vitro and in xenograft tumors demonstrated that cancer-associated mutations in the splicing regulator RBM10 switch its activity from tumor suppressor to an oncogene (Hernández et al, RNA Biology 2016). Based upon these mechanistic insights, antisense oligonucleotides (AONs) chemically modified to increase their selectivity and stability in vivo have been designed and tested in mouse models of lung adenocarcinoma with encouraging results (Hernández et al, in preparation). Alternative splicing profiling in melanoma showed that the pluripotency splicing regulator MBNL1 can contribute to tumor progression by targeting the DEK oncogene (Cifdaloz et al, submitted to Nature Comm). More generally, bioinformatic analyses and in vitro validation uncovered extensive networks of alternative splicing regulation in cancer (Sebestyén et al, Genome Research 2016). Work on reprogramming of B cells in mouse revealed key alternative splicing events and possible regulatory factors contributing to efficient reprogramming by the tarnscription factor CEBPa (Vivori et al, in preparation).

Overall good progress has been made, despite initial delays in RNA sequencing due to the need for sample optimization, data delivery and analysis, and due also to delays in the incorporation/replacement of some members of the team (see below). An unexpected conceptual complication emerged when a global picture of the effects of knock down of splicing factors on other components of the spliceosome was obtained: 50 - 60% of the knock downs affected the expression or alternative splicing of other splicing factors; while this introduces intrinsic complexity in the interpretation of the results of these genetic perturbations -making it necessary to adopt a novel conceptual framework for data analysis-, at the same time the results reveal a naturally existing, exquisitely interwoven, regulatory network which is one first major insight of our systems biology approach to alternative splicing.
Our RNA-seq datasets corresponding to the systematic knock down of each component of the splicing machinery represent the most comprehensive collection of available to systematically monitor the function of the components of this essential machinery and as such they can provide a unique resource to probe the function of the spliceosome in normal cellular differentiation and in disease, including cancer.
Along with mechanistic insights derived from detailed studies of the effects of mutations in splicing factors associated with cancer and genetic disease, including lung adenocarcinomas and retinitis pigmentosa, our results provide clues about critical genetic circuits behind the etiology of these pathologies and hint already at potential novel therapies, which are currently being tested in vitro as well as in animal models.