The aim of the first work (presented in the Chapter 2) was to investigate the role performed by SRPK2 kinase in the regulation of alternative splicing in SH-SY5Y neuroblastoma cells after paraquat treatment (a complex I mitochondrial respiratory chain inhibitor). Alternative splicing is a versatile form of genetic control whereby a common precursor messenger RNA (pre-mRNA) is processed into multiple mRNA isoforms differing in their precise combination of exon sequences. This process is particularly important in the nervous system and its essential nature is underscored by the finding that its misregulation is a common feature of human diseases, including neurodegenerative pathologies. Our approach to gain insight the regulation of neuron specific pre-mRNA splicing brought us to the characterization of SRPK2 kinase because this protein phosphorylates Serine/Arginine-rich domain (RS-domain)-containing proteins and it is expressed almost exclusively in the nervous system. In order to understand how SRPK2 intracellular localization and activity are regulated, in first instance we performed a mutational analysis. These mutants have been characterized by transient transfection in SH-SY5Y neuroblastoma cells. We analysed SRPK2 intracellular localization both under physiological condition and after generating stress through mitochondrial damage, since mitochondrial damage and oxidative stress are found in many neurodegenerative diseases. We also determined the effect of this stress treatment on SRPK2 phosphorylation and its nuclear translocation. We did this first by using the minigene E1A, an alternative splicing reporter system, and also by analysing both SR proteins intracellular localization and their phosphorylation status. This work showed that not only paraquat treatment increased the phosphorylated SRPK2 fraction, but also that a specific phosphorylation at its 581 residue could be connected with the nuclear translocation of SRPK2. Consequently, this nuclear translocation brought to a splicing change in the isoform ratio of the minigene reporter system. After the drug treatment we also observed a specific speckled enlarged pattern coupled with an increase in the phosphorylation level for the SR classical proteins, known targets of SR protein kinase 2. These findings supported a functional link between the nuclear translocation and the activity of this neuronal specific kinase. In the second line of our research (presented in the Chapter 3) we performed experiments assessing the function of the mammalian 3’ end processing factor CFIm68 in the mRNA export, thus confirming its action as an adaptor for TAP/NXF1 mRNA export receptor. In particular I helped to demonstrate that the tethering of CFIm68 promoted mRNA export by designing and performing an RNA FISH assay. I used a RNA-biotinylated probe that detected the intracellular localisation of an mRNA reporter construct co-transfected with the CFIm68 protein or control proteins. Therefore we observed an increase of the probe fluorescent cytoplasmic signal only in the presence of the overexpressed CFIm68 but not with other control proteins, observation confirmed by further Real Time PCR data. In the third line of our research (presented in the Chapter 4) we reported that CFIm68 was also involved in the 3’ end cleavage of mammalian histone transcripts (not polyadenylated) by interacting with the LSM11 U7 snRNP component both in vitro and in vivo thus increasing the efficiency of the 3’ end processing in vivo. In this context I performed the Bimolecular Fluorescence Complementation (BiFC) analysis. I co-transfected the CFIm68 and the LSM11 proteins (or its MPL loss-of-function mutant) fused respectively with the C-terminus and the N-terminus of a Venus-Yellow Fluorescent Protein. Thus I detected a nuclear fluorescent complementation in more than 90% of the cells (or not complementation with the MPL mutant counterpart). This data supported our whole characterized observation concerning the involvement of this mammalian cleavage factor in 3’ histone mRNAs processing.
(2010). New roles for RNA processing factors CFIm68 and SRPK2 highlight unexpected links in the control of mammalian gene expression. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).
BARABINO, SILVIA MARIA LUISA
|Data di pubblicazione:||30-mar-2010|
|Titolo:||New roles for RNA processing factors CFIm68 and SRPK2 highlight unexpected links in the control of mammalian gene expression|
|Settore Scientifico Disciplinare:||BIO/11 - BIOLOGIA MOLECOLARE|
|Scuola di dottorato:||Scuola di Dottorato in Medicina Traslazionale e Molecolare|
|Corso di dottorato:||MEDICINA TRASLAZIONALE E MOLECOLARE (DIMET) - 45R|
|Citazione:||(2010). New roles for RNA processing factors CFIm68 and SRPK2 highlight unexpected links in the control of mammalian gene expression. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2010).|
|Parole Chiave:||RNA processing, Paraquat, Neuroblastoma, Alternative Splicing, SR Proteins, SR Protein Specific Kinase 2|
|Appare nelle tipologie:||07 - Tesi di dottorato Bicocca post 2009|