Caracterización celular, bioquímica y funcional de TCERG1, un potencial factor de acoplamiento entre transcripción y splicing
- Autores: Miguel Sánchez Álvarez
- Directores de la Tesis: Carles Suñé Negre (dir. tes.)
- Lectura: En la Universidad Autónoma de Madrid ( España ) en 2009
- Idioma: español
- Tribunal Calificador de la Tesis: Luis Enjuanes (presid.), Isabel Correas Hornero (secret.), José Carlos Reyes Rosa (voc.), Juan Valcárcel Juárez (voc.), Susana de la Luna Gargantilla (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: Biblos-e Archivo
- Resumen
¿CARACTERIZACIÓN CELULAR, BIOQUÍMICA Y FUNCIONAL DE TCERG1, UN POTENCIAL FACTOR DE ACOPLAMIENTO ENTRE TRANSCRIPCIÓN Y SPLICING¿ Miguel Sánchez Álvarez Summary / Resumen en inglés The regulation of gene expression is critical for the maintenance of homeostasis in all organisms. The complexity of multicellular eukaryotes requires a versatile and tight gene expression regulation, which comprehends multiple stages, spatially and functionally organized in the cell nucleus.
Functional and physical communication among mRNP formation, maturation, packaging, surveillance and nucleocytoplasmic export mechanisms has been extensively reported. In the particular case of transcription and splicing, a bilateral Communications between both processes is believed to constitute a regulatory mechanism per se for the appropriate tuning of gene expression, especially through the modulation of the alternative processing of pre-mRNAs. This thesis report documents our contribution to the characterization and further understanding of the eukaryotic factor TCERG1 (previously named CA150), that could participate in the establishment and regulation of those communication events. We studied the subnuclear distribution of TCERG1 by optical microscopy and found that this factor constitutively accumulates in the speckles, nuclear structures enriched in mRNA methabolism-related components. TCERG1 localizes within the transcriptionally active periphery of these compartments. Our studies indicated that the carboxylterminal portion of TCERG1 (aa 591-1098) and potentially its FF5 domain (aa 956-1004) are important for this association. Biochemical analysis of the in vivo interactions established by this protein suggested that TCERG1 specifically interacts with transcription elongation regulators and spliceosomal components, and that multiple structural domains may be simultaneously required for the stability of these interactions. Immunodepletion of TCERG1 from standard HeLa nuclear extracts had no significant effect on their constitutive splicing activity in vitro; however, transient overexpression of TCERG1 specifically influenced the alternative processing pattern of the fibronectin ED-I exon in vivo. This effect was independent of the promoter used, and required the integrity of WW domains 1 and 2 as well as the carboxylterminal portion of TCERG1.
Finally, we have characterized one of the posttranslational modifications that potentially regulate TCERG1 function: SUMO conjugation. This protein is a bona fide SUMOylation substrate in vivo and can be potentially modified by all three SUMO paralogs. We identified two lysine residues within its aminoterminal sequence, K503 and K608, as major in vivo SUMO acceptors. Their surrounding sequences largely fit in with the recently proposed consensus NDSM motif !KxE/D(D/Ex)n, and their features and relative location within the structural organization of TCERG1 are very conserved among most metazoan homologs. Blockade of these motifs by K to R substitution had no apparent effect on subcellular localization nor on its negative influence on ED-I exon processing; however, TCERG1 mutants exhibited increased transcription transactivation activity when tested in two independent experimental systems, suggesting a negative modulatory role for these residues in the transcription-related activity of TCERG1.
