Objectives

The major goal of this work is to learn the molecular mechanisms of splice site pairing, the cross-intron communication of the intron ends and thereby to understand more about the architecture of early spliceosomal complexes. When this work began, not much was known about the communication of the splice sites during early spliceosome assembly.

The U2 snRNP seemed to play a central role in splice site recognition/pairing during the early stages of the splicing process since it was functionally and loosely associated with the spliceosomal E complex (Das et al., 2000). However, the organization of U2 relative to the functional sites of the pre-mRNA and to the U1 snRNP is not known. We were interested to know the proximities among the components of the early spliceosomal complexes especially the U1 and U2 snRNPs. In order to investigate the RNA proximities of U2 relative to the functional sites of the pre-mRNA and to the U1 snRNP, we decided to use the site-directed hydroxyl radical probing method. Therefore, we have attached the hydroxyl radical probe Fe-BABE to the 5' end of the U2 snRNA and analyzed the proximities to pre-mRNA and the U1 snRNA in E and A spliceosomal complexes.

For this purpose, the endogenous U2 were depleted from Hela nuclear extract and Fe-BABE-modified U2 snRNPs were provided to the sytem. Therefore, we used the two-step reconstitution/complementation system initially developed by Segault et al. (1995).

The two steps of the system are as follows. First, core U2 snRNPs were reconstituted in vitro from anti-m3G purified native snRNP proteins (total proteins) and U2 snRNA.

Second, the core U2 snRNP was added to HeLa cell nuclear extract depleted of U2 snRNP but still containing particle-specific proteins. These particle-specific proteins were allowed to assemble onto 12S U2 snRNPs to form 17S U2 snRNPs. By adding pre-mRNA, the spliceosome assembly and the splicing reaction were analyzed.

It was known that, in contrast to other snRNAs, in vitro transcribed U2 snRNA without any modifications did not support splicing (see 2.1.2.1.1). To generate Fe-BABE modified U2 snRNAs, we needed to identify which modified residues are required for U2 function in splicing so that the chimaeric U2 snRNA with the required modifications and the Fe-BABE could be prepared. Thus, the results of the first part of this work paved the way for the analysis of the question in the second part.

In the first part of this thesis, the role and the requirement of the individual modification of U2 snRNA within the first 24 nucleotides were studied. At the beginning of this work, it was known that the in vitro transcribed, unmodified U2 snRNA was not capable of complementing pre-mRNA splicing in HeLa cell nuclear extracts depleted of U2, while the native, HeLa U2 snRNA did so efficiently (Segault et al., 1995). This indicated for the first time that modified nucleotides of U2 snRNA are essential for its function. Then, in a study carried out in Xenopus oocytes, it was shown that the modifications within the first 27 nucleotides of U2 snRNA were required for splicing and spliceosome assembly in vivo (Yu et al., 1998). However, the role that these modified nucleotides (13 pseudouridines and 10 2'-O-methyl groups) play for the structure and function of U2 snRNA remained to be investigated. In addition to this, according to Yu et al., 1998, the m3G cap of U2 snRNA was found to be essential for splicing within the context of the experimental system used. Therefore, we set out to investigate the requirement and the role of individual modifications within the first 24 nucleotides of the U2 snRNA by using the two-step reconstitution/complementation system. The native HeLa U2 snRNA, in vitro transcribed U2 snRNA and the chimaeric U2 snRNAs were used in this system. The chimaeric U2 snRNAs which contain different numbers and types of modifications within the first 24 nucleotides of U2 snRNA were generated via Moore and Sharp ligation (Moore and Sharp, 1992). After we have shown that the three pseudouridines and five 2'-O-methylations within the first 24 nucleotides of U2 snRNA are required for splicing, we analyzed the step of spliceosome assembly that they are essential for.

Importantly, it was also demonstrated in this study that the 5' terminal m3G cap is not required for the function of U2 snRNA in splicing in vitro. These findings were of utmost

practical importance, as this facilitates significantly the chemical synthesis of 5' terminal U2 RNA oligonucleotide which is 24 nucleotide long and contain the required modifications and a SH group at its 5' end. This SH groups is necessary to attach hydroxyl radical generator, Fe-BABE, at the 5' end of U2 snRNA to investigate which RNAs are in close proximity to this region of U2 snRNA in early spliceosomal complexes.

Distinct signals were observed on the pre-mRNA and U1 RNA in E complex indicating that these regions are located in close proximity to the 5' end of U2 RNA as investigated by primer extension analysis. Furthermore, the spatial proximities of the RNAs upon addition of ATP (the spliceosomal A complex) were investigated. In this way, the conformational changes of U1 and pre-mRNAs during spliceosomal E to A complex transition were monitored.

The cleavages observed on U1 snRNA provided constraints for modeling the orientation of U1 and U2 snRNPs in the E complex by using the protein-free 3D structure of U1 snRNA and the cryo-EM structure of U1 snRNP (Stark et al., 2001). Mapping of the cleavages observed on U1 RNA onto these structures revealed a distinct orientation of U2 relative to U1 in early spliceosomal complexes. The 5' end of U2 appears to be located on a specific "side" of the U1 snRNP, which for historical reasons designated as the "back"

of the particle. By the help of the hydroxyl radical cleavage data on U1, the 5' end of U1 snRNA was placed on the U1 snRNP structure.