Investigation of the higher order structure of the spliceosomal RNA network
Başlık çevirisi mevcut değil.
- Tez No: 401188
- Danışmanlar: PROF. DR. RALF FICNER, PROF. DR. REINHARD JAHN
- Tez Türü: Doktora
- Konular: Tıbbi Biyoloji, Medical Biology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2006
- Dil: İngilizce
- Üniversite: Georg-August-Universität Göttingen
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 187
Özet
Özet yok.
Özet (Çeviri)
The removal of introns from pre-mRNA is achieved by a highly dynamic ribonucleoprotein complex termed the spliceosome. It is formed by the ordered association of the U1, U2, U4/U6 and U5 snRNPs (small nuclear ribonucleoproteins) and non-snRNP proteins with conserved regions of the pre-mRNA at the 5' and 3' splice sites, and the so-called branch site. Spliceosome assembly begins with the association of U1 and U2 to form the E complex. Subsequently, the A complex is formed upon stable integration of the U2 snRNP and is then converted to the B complex by association of the tri-snRNP (U4/U6.U5). After rearrangements, the catalytically active C complex is formed and the splicing reaction ensues. Recognition and pairing of the correct 5' and 3' splice sites (ss) of a pre-mRNA are critical events that occur early during spliceosome assembly. Little is known about the spatial organization in early spliceosomal complexes of the U1 and U2 snRNPs, which together with several non-snRNP proteins, are involved in juxtapositioning the functional sites of the pre-mRNA. To better understand the molecular mechanisms of splice site recognition/pairing, we have examined the organization of U2 relative to U1 and premRNA in spliceosomal complexes via site-directed hydroxyl radical probing. For this purpose, the hydroxyl radical generator, Fe-BABE, was tethered to U2 snRNA and Fe- BABE modified U2 snRNPs were reconstituted in vitro, and used to complement splicing reactions lacking U2. To generate Fe-BABE modified U2 snRNAs active in splicing, we first determined which modifications of the U2 snRNA are essential and what role they play in U2 snRNP assembly and/or splicing. U2 snRNA has a 5'-terminal m3G cap and many internal modifications including ten 2'- O-methylated residues and thirteen pseudouridines. At the onset of this work, it was known that modifications within the first 27 nucleotides of U2 snRNA, including the m3G cap, are required for splicing and/or U2 snRNP biogenesis in Xenopus oocytes. However, precisely which modifications of U2 snRNA are essential and their role in snRNP and/or spliceosome assembly in HeLa nuclear extract was not clear. To address these questions, chimaeric U2 snRNAs were synthesized via Moore and Sharp ligation containing different numbers and types of modifications within the first 24 nucleotides of U2 snRNA and assayed for activity. It was shown that the three pseudouridines and five 2'-O-methyl groups within the first 24 nucleotides of U2 snRNA, but not the m3G cap, are required for efficient pre-mRNA splicing in vitro. The in vitro assembly of 17S U2 snRNPs was not dependent on the presence of modified U2 residues. However, spliceosomal complex formation assays demonstrated that internal modifications at the 5' end of U2 snRNA are required for the formation of the ATP-independent, early spliceosomal E complex. This data suggested that modifications within the first 24 nucleotides of U2 snRNA might play a role in facilitating the interaction of U2 with U1 snRNP and/or other factors within the E complex. In the second part of this work, RNA proximities within the E and A spliceosomal complexes were analyzed by site-directed generation of hydroxyl radicals from Fe-BABE covalently attached to the U2 snRNA. Fe-BABE was attached to the 5' terminal nucleotide, which was inserted during the chemical synthesis of the 24 nucleotide long oligomer comprising the 5' end of U2 snRNA with all required natural modifications. Significantly, 5' Fe-BABE-U2 snRNPs supported both spliceosomal assembly and splicing in vitro. Hydroxyl radical probing with 5' Fe-BABE U2 snRNPs demonstrated that the functional sites of the pre-mRNA (the 5' and 3' splice sites, the anchoring sequence and the polypyrimidine tract) are in close proximity to the 5' end of U2 in the E complex, the earliest detectable splicing complex. In A complex, the proximities to the pre-mRNA were generally similar. Interestingly, U1 snRNA was found to be in close proximity to the 5' end of U2 in both E and A complexes. Upon A complex formation, the relative orientation of U1 snRNP to U2 snRNP did not change. This work showed for the first time that, as early as in the E complex, the U2 and U1 snRNPs are in close proximity and in a fixed orientation relative to one another. As such, they“bridge”the 5' and 3' ends of the pre-mRNA. This particular orientation of U1 relative to U2 is dependent on active spliceosomal complex formation on pre-mRNA, and on a functional 5' splice site. By mapping the U1 snRNA cleavage data onto the protein-free 3D U1 snRNA structure, it was shown that the cleaved nucleotides are oriented such that they could face a single point source of hydroxyl radicals, consistent with the idea that the 5' end of U2 is located on one side of the U1 snRNA. When the cleavage data on U1 snRNA were mapped onto the U1 snRNA in the U1 snRNP model, cleaved nucleotides were still found on one side of the U1 snRNA at the“back”of the U1 snRNP. These data indicate that the U2 snRNP interaction domain is located predominantly at the“back”of the U1 snRNP and, furthermore, support the apparent structure/position of the U1 snRNA in the U1 snRNP 3D model. The detection of hydroxyl radical cleavages at the 5' end of the U1 snRNA suggests that this functionally important region is also positioned on the same side of the U1 snRNP as the other cleaved nucleotides. These studies reveal that functional sites of the pre-mRNA and U1 snRNA are located close to the 5' end of U2 both in E and A complexes. U1 and U2 snRNPs may form a molecular bridge between the ends of the intron, much like the U11/U12 di-snRNP. The results of this work thus suggest that a major rearrangement in the 5' end of U2 relative to the reactive functionalities of the pre-mRNA might not be required to generate catalytically active spliceosomes in the subsequent steps leading to the first step of splicing.
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