The aim of the research that is being conducted in our Laboratory is, on one hand, to decipher the mechanisms involved in biogenesis of messenger RNA (mRNA) and, on the other, to contribute to a better understanding of the pathogenesis of diseases caused by genetic errors that affect this process.
We have shown that splicing factors U2AF65 and U2AF35 shuttle continuously between the nucleus and the cytoplasm by a mechanism that involves carrier receptors and is independent from binding to mRNA. Nucleo-cytoplasmic shuttling may represent a means to control the availability of splicing factors in the nucleus to initiate spliceosome assembly and therefore contribute to regulate splicing. Alternatively, U2AF may associate with spliced mRNPs and participate in coupling splicing to later events in the life of mRNA.
We have also shown that vertebrate transcripts coding for U2AF35 are alternatively spliced, and we are currently using RNA interference analysis to identify pre-mRNA targets regulated by each isoform. We have established and further developed in the lab photobleaching microscopy methodologies and we are currently applying these techniques to study the dynamics of spliceosome assembly in live cells.
We have also established cellular model systems to visualise by light microscopy the recruitment of the transcription and RNA processing machinery onto nascent transcripts. We found that mutant β-globin mutant pre-mRNAs that fail to be spliced are not exported to the cytoplasm and remain stalled at the site of transcription. The mutant transcripts do not recruit spliceosomes or export factors and we plan to further dissect the molecular mechanisms involved in this RNA surveillance process.
- Heyd, F., Carmo-Fonseca, M., Moroy, T. (2008). Differential isoform expression and interaction with the P32 regulatory protein controls the subcellular localization of the splicing factor U2AF26. Journal of Biological Chemistry 2008 May 6. [epub ahead of print]
- de Almeida, S.F., Carmo-Fonseca, M. (2008). The CTD role in cotranscriptional RNA processing and surveillance. FEBS Letters 2008 Apr 22. [epub ahead of print]
- Rino, J., Desterro, J.M., Pacheco, T.R., Gadella, T.W. Jr, Carmo-Fonseca, M. (2008). Splicing factors SF1 and U2AF associate in extraspliceosomal complexes. Molecular and Cellular Biology 28(9), 3045-57.
- Carneiro, T., Carvalho, C., Braga, J., Rino, J., Milligan, L., Tollervey, D., Carmo-Fonseca, M. (2008). Inactivation of cleavage factor I components rna14p and rna15p induces sequestration of small nucleolar ribonucleoproteins at discrete sites in the nucleus. Molecular Biology of the Cell 19(4), 1499-508.
- Carmo-Fonseca, M., Carvalho, C. (2007). Nuclear organization and splicing control. Advances in Experimental Medicine and Biology 623, 1-13.
- Carmo-Fonseca, M. (2007). How genes find their way inside the cell nucleus. Journal of Cell Biology 179(6), 1093-4.
- Rino, J., Carvalho, T., Braga, J., Desterro, J.M., Lührmann, R., Carmo-Fonseca, M. (2007). A stochastic view of spliceosome assembly and recycling in the nucleus. PLoS Computational Biology 3(10), 2019-31.
- Custódio, N., Vivo, M., Antoniou, M., Carmo-Fonseca, M. (2007). Splicing- and cleavage-independent requirement of RNA polymerase II CTD for mRNA release from the transcription site. Journal of Cell Biology 179(2), 199-207.
- Carneiro, T., Carvalho, C., Braga, J., Rino, J., Milligan, L., Tollervey, D., Carmo-Fonseca, M. (2007). Depletion of the yeast nuclear exosome subunit Rrp6 results in accumulation of polyadenylated RNAs in a discrete domain within the nucleolus. Molecular and Cellular Biology 27(11), 4157-65.
- de Almeida, S.F., Fleming, J.V., Azevedo, J.E., Carmo-Fonseca, M., de Sousa, M. (2007). Stimulation of an unfolded protein response impairs MHC class I expression. Journal of Immunology 178(6), 3612-9. PMID: 17339458 [PubMed - indexed for MEDLINE]
- Braga, J., McNally, J.G., Carmo-Fonseca, M. (2007). A reaction-diffusion model to study RNA motion by quantitative fluorescence recovery after photobleaching. Biophysical Journal 92(8), 2694-703.
- Rino, J., Carvalho, T., Braga, J., Desterro, J. M. P., Lührmann, R., and Carmo-Fonseca, M. (2007). A stochastic view of spliceosome assembly and recycling in the nucleus PLoS Computational Biology 3, 2019-2031.
- Barbosa-Morais, N., Carmo-Fonseca, M., Aparicio, S. (2006). Systematic genome/wide annotation of spliceosomal proteins reveals differential gene family expansion. Genome Research 16, 66-77.
- Gama-Carvalho, M., and Carmo-Fonseca, M. (2006). Heterokaryon assays. In "Cell Biology – a Laboratory Manual", ed. Julio Celis, Elsevier Science USA.
- Custodio, N., Carvalho, C., Carneiro, T. and Carmo-Fonseca, M. (2006). In situ hybridization for simultaneous detection of DNA, RNA and protein. In "Cell Biology – a Laboratory Manual", ed. Julio Celis, Elsevier Science USA.
- Kozlova, N., Braga, J., Lundgren, J., Rino, J., Young, P., Carmo-Fonseca, M. (2006). Studies on the role of NonA in mRNA biogenesis. Experimental Cell Research 312, 2619-30.
- Custódio, N., Antoniou, M., Carmo-Fonseca, M. (2006). Abundance of the largest subunit of RNA polymerase II in the nucleus is regulated by nucleo-cytoplasmic shuttling. Experimental Cell Research 312, 2557-67.
- Pacheco, T.R., Moita, L.F., Gomes, A.Q., Hacohen, N., Carmo-Fonseca, M. (2006). RNAi Knockdown of hU2AF35 Impairs Cell Cycle Progression and Modulates Alternative Splicing of Cdc25 Transcripts. Molecular Biology of the Cell 17(10), 4187-4199.
- Pacheco, T.R., Coelho, M.B., Desterro, J.M.P., Mollet, I., and Carmo-Fonseca, M. (2006). In vivo requirement of the small subunit of U2AF for recognition of a weak 3’ splice site. Molecular Cellular Biology 26(21), 8183-90.
- Mollet, I., Barbosa-Morais, N.L., Andrade, J., Carmo-Fonseca, M. (2006). Diversity of human U2AF splicing factors. FEBS Journal 2006 Oct;273(21), 4807-16.
- Gama-Carvalho, M., Barbosa-Morais, N.L., Brodsky, A.S., Silver, P.A., Carmo-Fonseca, M. (2006). Genome wide identification of functionally distinct subsets of cellular mRNAs associated with two nucleocytoplasmic-shuttling mammalian splicing factors. Genome Biology 7(11), R113.
- Pacheco, T.R., Gomes, A.Q., Barbosa-Morais, N.L., Benes, V., Ansorge, W., Wollerton, M., Smith C.W., Valcarcel, J., and Carmo-Fonseca, M. (2004). Diversity of vertebrate splicing factor U2AF35: identification of alternatively spliced U2AF1 mRNAs. Journal of Biological Chemistry 279, 27039-27049.
- Custódio, N., Carvalho, C., Condado, I., Antoniou, M., Blencowe, B.J. and Carmo-Fonseca, M. (2004). In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei. RNA 10, 622-633.
- Braga, J., Desterro, J.M., Carmo-Fonseca, M. (2004). Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes. Molecular Biology of the Cell 15, 4749-4760.
- Calapez, A., Pereira, H.M., Calado, A., Braga, J., Rino, J., Carvalho, C., Tavanez, J.P., Wahle, E., Rosa, A.C., and Carmo-Fonseca, M. (2002). The intranuclear mobility of messenger RNA binding proteins is ATP-dependent and temperature-sensitive. Journal of Cell Biology 159, 795-805.
- Carmo-Fonseca, M. (2002). The contribution of nuclear compartmentalization to gene regulation. Cell 108, 1-20.
Key lab techniques: confocal fluorescence microscopy, FISH, immunofluorescence and GFP-fusion protein analysis, FRAP, FRET, RNAi.
Key lab reagents: GFP-constructs, antibodies, cell lines.
Lab contact: Joana Desterro: Joanadesterro@fm.ul.pt
Lab website: www.imm.ul.pt