1. How is gene expression modulated by interactions between transcription and RNA processing?

An increasingly prominent concept suggests that coordination between transcription and RNA processing allows fine-tuning of gene outputs. Over the years, major advances in understanding coupled gene expression events have emerged from new technologies. In collaboration with the Proudfoot lab at Oxford University, our group pioneered methodologies for the isolation and analysis of newly transcribed RNAs associated with RNA polymerase II. Few labs around the world work on nascent RNA because it corresponds to a very small fraction of total cellular RNA and therefore presents a purification challenge. Consequently, our lab stands uniquely positioned to elucidate the network of interactions involved in the early steps of gene expression. Our previous analysis of intact nascent RNAs using long-read sequencing showed that although many transcripts are immediately spliced, a significant fraction of nascent pre-mRNAs remained unspliced and failed to be cleaved at the polyA site (PMID: 33735606). What happens to these unspliced and uncleaved nascent transcripts? Do they eventually undergo splicing, polyadenylation and export to the cytoplasm, or do they represent dead-end products destined for degradation in the nucleus? Why are some pre-mRNAs rapidly spliced while others remain unspliced? Is delayed splicing functional? We aim to address these questions by tracking in space and time newly synthesized pre-mRNAs and their interactions with the transcription machinery.

2. How mutations that disrupt splicing of BRCA transcripts lead to hereditary cancer?

Breast cancer is the most common cancer in women, and up to 10% of breast cancer patients are genetically predisposed. Hereditary breast and ovarian cancer syndrome (HBOC) is a disorder with a huge impact on affected families, as cancers often develop in women at childbearing age. We have the ambition of contributing to HBOC prevention by enabling the understanding of how germline mutations that disrupt splicing of BRCA1 and BRCA2 transcripts interfere with cellular homeostasis favoring oncogenesis. A wide spectrum of deleterious BRCA1 and BRCA2 mutations have been identified, the vast majority of which result in protein loss-of-function. We are focusing on the BRCA2 founder mutation (c.156_157insAlu) that is present in approximately 30% of all Portuguese families with HBOC, representing 55% of all BRCA2 germinal mutation carriers in Portugal. The mutation consists in an Alu insertion that leads to full in-frame skipping of exon 3. Although it is generally assumed that this mutation leads to expression of a shorter BRCA2 protein lacking amino acids encoded by exon 3, very little is known about the molecular pathways altered in cells harboring this mutation. Due to the fact that the BRCA2 protein is critical for the protection of nascent strands at stalled replication forks and the repair of DNA double-strand breaks by homologous recombination, and estrogen stimulates both transcription and cell proliferation, we hypothesize that in epithelial stem/progenitor cells harboring loss-of-function BRCA2 mutations, estrogen will increase transcription-coupled replicative stress affecting stem cell fate.

3. Can fundamental knowledge of splicing mechanisms be translated into improved splicing modulation therapies?

Emerging therapies that are either RNA-based or RNA-targeting molecules are being rapidly developed. RNA therapies have the potential to improve the lives of many people affected by difficult-to-treat diseases by selectively acting on hitherto “undruggable” cellular components. Namely, the splicing modifying drug Nusinersen had a transformative impact in the treatment of infants with spinal muscular atrophy (PMID: 30221755).

Our goal is to harness the potential of RNA therapies for heart diseases. Building on recent evidence suggesting that RNA splicing plays an important role in the pathogenesis of familial hypertrophic cardiomyopathy (HCM), which is the most common inherited heart disease and a leading cause of sudden cardiac death, we are investigating the impact of HCM-associated mutations on RNA splicing, using as a model system cardiomyocytes differentiated in vitro from patient-derived iPSCs (iPSC-CMs). We are particularly interested in mutations located in introns, which may interfere with splicing but are often disregarded in conventional medical genetic diagnosis (PMID: 28497172). Our lab has generated a collection of iPSCs harboring mutations that affect splicing of HCM-associated genes by distinct mechanisms. We use cardiomyocytes differentiated from these iPSCs as cellular models to develop splicing correction approaches. Namely, we are designing antisense oligonucleotides (ASOs) to inactivate cryptic splice sites created by the mutations, and we are collaborating with chemical biologists from the University of Copenhagen to explore strategies for effective delivery of ASOs into cardiomyocytes. Additionally, we are exploring Cas13-mediated RNA editing for splicing correction of mutant cells. We further hypothesize that by elucidating the spatial and temporal organization of RNA splicing within the nucleus we can pioneer innovative strategies to develop clinically impactful splicing modulation therapies.

The 4D organization of pre-mRNA splicing

Cardiac Splicing as a Therapeutic Target

Impact of the Portuguese BRCA2 founder mutation on transcription and tissue stem cell identity

Lima BA, Pais AC, Dupont J, Dias P, Custódio N, Sousa AB, Carmo-Fonseca M, Carvalho C. Genetic modulation of RNA splicing rescues BRCA2 function in mutant cells. Life Sci Alliance. 2024;8(3):e202402845.

Jager J, Ribeiro M, Furtado M, Carvalho T, Syrris P, Lopes LR, Elliott PM, Cabral JMS, Carmo-Fonseca M, da Rocha ST, Martins S. Patient-derived induced pluripotent stem cells to study non-canonical splicing variants associated with Hypertrophic Cardiomyopathy. Stem Cell Res. 2024;81:103582.

Gotthardt M, Badillo-Lisakowski V, Parikh VN, Ashley E, Furtado M, Carmo-Fonseca M, Schudy S, Meder B, Grosch M, Steinmetz L, Crocini C, Leinwand L. Cardiac splicing as a diagnostic and therapeutic target. Nat Rev Cardiol. 2023;20(8):517–530.

Barbosa P, Savisaar R, Carmo-Fonseca M, Fonseca A. Computational prediction of human deep intronic variation. GigaScience. 2023;12:giad085.

Sousa-Luis R, Dujardin G, Zukher I, Kimura H, Carmo-Fonseca M, Proudfoot NJ, Nojima T. POINT Technology illuminates the processing of polymerase-associated intact nascent transcripts. Mol Cell. 2021;81(9):1935–1950.

Prudêncio P, Savisaar R, Rebelo K, Martinho RG, Carmo-Fonseca M. Transcription and splicing dynamics during early Drosophila development. RNA. 2021;28:1–23.

Nojima T, Rebelo K, Gomes T, Grosso AR, Proudfoot NJ, Carmo-Fonseca M. RNA Polymerase II phosphorylated on CTD Serine 5 interacts with the spliceosome during co-transcriptional splicing. Mol Cell. 2018;72(2):369–379.

Bernardes de Jesus B, Matinho SP, Barros S, Sousa-Franco A, Alves-Vale C, Carvalho T, Carmo-Fonseca M. Silencing of the lncRNA Zeb2-NAT facilitates reprogramming of aged fibroblasts and safeguards stem cell pluripotency. Nat Commun. 2018;9(1):94.

Vaz-Drago R, Custódio N, Carmo-Fonseca M. Deep intronic mutations and human disease. Hum Genet. 2017;136:1093–1111.

Nojima T, Gomes T, Grosso AR, Kimura H, Dye MJ, Dhir S, Carmo-Fonseca M, Proudfoot N. Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing. Cell. 2015;161(3):526–540.

A complete list of publications can be found here.

Carmo-Fonseca received the Gulbenkian Science Award (Fundação Calouste Gulbenkian, Portugal) in 2007, the “Prémio Pessoa” (Portuguese award in Culture, Art and Sciences) in 2010, and the University of Lisbon Award in 2023

https://orcid.org/0000-0002-3402-7143

https://scholar.google.com/citations?user=KbjLqH0AAAAJ&hl=en