Investigation of the Mechanisms of Action of Exonic splicing Enhancers Using Single Molecule Methods

The main purpose of studying the state of binding SRm160 at single molecule level is to extract fundamental information about this protein (SRm160) and the mechanism of its involvement in splicing reactions. Single molecule methods have been widely used in colocalisation applications due to their advantages in following the state of a specific molecule (single molecule) of interest in real time. In the context of SRm160 protein, its involvement in splicing reaction and its relationship with other snRNPs (factors) remain important issues that may be involved in the early complex E and complex A, such that, it may induce a restructuring of the pre-mRNA complexes. Very few research papers were published regarding what is SRm160. Since then, it was believed that the binding of this protein to RNA is through other SR proteins as SRm160 does not consist of RRM motif domain. It was also believed that SRm160 binds as a bridge along side RNA. Regarding all studies on SRm160, we found it interesting to study the state/ the behaviour of this protein at single molecule level in order to elucidate its mechanism in real time. This thesis identified new insight for how SRm160 protein interact with RNA and how other snRNPs might affect this reaction. This identification is based on single molecule technique that depend on counting bleaching steps of the attached fluorescent fluorophores (colocalisation measurements). Additionally, the project was extended to initiate a FRET analysis in order to investigate whether the bound-SRm160 a affected the flexibility movement of a substrate or not. Two different approaches are pursued: a. The fifth chapter proposes a novel single molecule technique which is introduced as single molecule colocalization system. This system is using a suitable microscope for excitation via total internal reflection (TIRF) to obtain the single molecule data. Two different fluorophores (the chosen fluorophores are mEGFP and Cy5) were implemented to allow the investigation of the interaction between SRm160 molecules and the premRNA during splicing reaction. Multiple pre-mRNAs were used, such as, GloC and SMN2 constructs. Multiple conditions were tested, such as, the absence of ATP, the presence of ATP and the presence of other factors (anti oligonucleotides) which might affect the complexes formation or even snRNPs binding. Following this, we can propose a new paradigm for how SRm160 interacts with pre-mRNA. The obtained results demonstrate that the association of GFP-SRm160 with SMN2 substrate is increased in the presence of the ESEs site. This association is not affected in the presence of anti-U1 oligonucleotide. The distribution of this association is significantly reduced with a single SRm160 protein remaining on the substrate in the presence of anti-U2 snRNP. These results demonstrate various points; the association of SRm160 with SMN2 pre-mRNA is enhanced via ESE sites, and is highly dependent on U2 snRNP but not U1 snRNP. These results refute the speculation in the literature that the association of SRm160 is highly dependent on U1 snRNP and stabilized by U2 snRNP. b. The sixth chapter of the thesis proposes FRET system in order to allow deeper investigation regarding the relationship between pre-mRNA and SRm160 in a real time, such as, the state of fluctuations of a molecule of interest (forked DNA or double-labelled RNA) in the presence of SRm160. This chapter is motivated by the previous contribution which identified the state of binding of SRm160 during splicing reaction. The obtained results showed that (1) No FRET signal was appears at the condition supporting the formation of the early complex E. This supports the colocalisation results which indicates a multiple binding of SRm160 molecules alongside the pre-mRNA substrate. This means that the presence of a multiple number of the large molecule (SRm160) prevents the energy transfer. Additionally, this result could be a consequence of limited conformational fluctuations caused by the presence of this rigid molecule. -(2) FRET signal was appeared at the condition supporting the formation of complex A. This finding is also consistent with the colocalisation results at the same condition. Phosphorylation decreased the number of SRm160 proteins. This means that energy transfer between the two flurophores was possible. -(3) The presence of anti-U1 oligo prevented the FRET signal to be detected. This means that SRm160 remained bound to the construct. The persistence of a large molecule a affected the FRET signal as identified at the early complex E condition. This finding confirms our conclusion that the binding of SRm160 does not depend on the presence of U1 snRNP. In all of the proposed methods an automatic system was available to reduce the time required to achieve one experiment. However, to compute results we are looking for, for example, to compute the number of molecules regarding each step, calculation has to done manually for each file of date. The manual processes are time consuming and might lead to an unintended errors. To overcome the manual limitations, the proposed methods are then evaluated for data analysis and data documentation.