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Investigating the Assembly of Ribonucleoprotein complexes (RNPs)
Eukaryotic cells contain numerous small nuclear ribonucleoproteins (RNPs) that function in different RNA-processing events in the nucleus. The spliceosomal U1, U2, U5 and U4/U6 snRNPs (small nuclear RNPs) make up one class of complexes that are catalysing a well defined process called pre-mRNA splic...
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| Format: | Dissertation |
| Sprache: | Englisch |
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Philipps-Universität Marburg
2007
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| Zusammenfassung: | Eukaryotic cells contain numerous small nuclear ribonucleoproteins (RNPs) that function in different RNA-processing events in the nucleus. The spliceosomal U1, U2, U5 and U4/U6 snRNPs (small nuclear RNPs) make up one class of complexes that are catalysing a well defined process called pre-mRNA splicing. Pre-mRNA splicing is the process by which non-coding regions (introns) are removed from pre-mRNA transcripts and the protein coding elements (exons) assembled into mature mRNAs before the RNA leaves the nucleus. Another class comprises the box C/D snoRNPs (small nucleolar RNPs) that are involved in processing of the ribosomal RNA (rRNA) in the nucleolus. These complexes function as guides in the site-specific 2' O-methylation of riboses in the precursor rRNA as well as assisting in rRNA biogenesis.
Though the above-named U4/U6 snRNP and the box C/D snoRNP complexes are distinct in both composition and function, they share a similar RNA component and protein composition. Their RNA component can form a so called kink-turn (k-turn) motif (in the U4 5′ stem-loop and box C/D and B/C motif in box C/D snoRNAs), which is bound by a 15.5 kilodalton protein (15.5K).
Besides protein 15.5K, the U4/U6 snRNP and the box C/D snoRNP complexes possesses several complex-specific proteins. In addition to seven Sm and seven LSm proteins that bind the Sm site of the U4 snRNA and the 3' end of U6 snRNA respectively, four proteins have been found to be associated with the human U4/U6 snRNP. These include the hPrp31 protein (also called 61K) and three proteins with molecular weights of 20, 60 and 90 kDa that form a biochemically stable, hCypH/hPrp4/hPrp3 (also 20/60/90K) protein complex. As well the box C/D snoRNPs are associated with a number of complex-specific proteins, which practically perform processing of the rRNA. The box C/D snoRNPs, like the U8 and U14 box C/D snoRNA, have been shown to bind proteins NOP56, NOP58, TIP48, TIP49 and fibrillarin. Lastly, the 15.5K protein and the hU3–55K protein belong to the complex assembled on the U3 box B/C motif of the U3 box C/D snoRNA.
The assembly of the RNP complexes is a multiple-stage process that is initiated by 15.5K protein binding to the respective k-turn motif. Protein 15.5K is unique in that it is essential for the hierarchical assembly of the above-named three RNP complexes. Protein 15.5K interacts with the cognate RNAs via an induced-fit mechanism, which results in the folding of the surrounding RNA to create binding site(s) for the RNP-specific proteins. It has been shown that protein-RNA interactions are essential for the binding of complex-specific proteins to the U4/U6 snRNP, C/D snoRNP, and the RNP complex assembled on the U3 box B/C motif. However, at the beginning of this work it was unknown whether protein 15.5K also mediates RNP formation through direct protein-protein interactions with the complex-specific proteins. To investigate this possibility, a series of protein 15.5K mutations were created in which the surface properties of the protein had been changed. Within the scope of this work, their ability to support the formation of the three distinct RNP complexes was assessed and the formation of each RNP was found to require a distinct set of regions on the surface of the 15.5K protein. This implies that protein-protein contacts are essential for RNP formation in each complex. Further supporting this idea, direct protein protein interaction was observed between hU3 55K and 15.5K. In conclusion, the data obtained suggest that the formation of each RNP involves the direct recognition of specific elements in both 15.5K protein and the specific RNA.
The U4/U6 snRNP-specific protein hPrp31 and the box C/D snoRNP-specific proteins NOP56 and NOP58 have been shown to be homologous, sharing a conserved domain, the so-called Nop domain. At the beginning of this work, it was unclear how protein hPrp31 and proteins NOP56/NOP58 assemble specifically onto the U4/U6 snRNP and box C/D snoRNPs, respectively. To address this question, structural requirements for the association of protein hPrp31 with the U4 snRNP in vitro were analysed. By employing point and deletion mutants of the U4 snRNA, structural features within the U4 snRNA necessary for binding of protein hPrp31 to the U4/U6 snRNP were determined. The findings indicate that the specificity of hPrp31 binding is provided by stems I and II of the U4 snRNA and suggest a way in which protein hPrp31 and proteins NOP56/NOP58 may specifically assemble onto the U4/U6 snRNP and box C/D snoRNPs, respectively. Furthermore, the results presented here provide evidence that the Nop domain on its own is necessary and sufficient for hPrp31 binding to the U4 snRNP, thus suggesting that this domain may be a novel RNA-binding domain.
Recently it has been shown that protein hPrp31 is also of clinical interest. Two mutations (A194E, A216P) in the human hPrp31 gene (PRPF31) are correlated with the autosomal dominant form of retinitis pigmentosa, a disorder that leads to degeneration of the photoreceptors in the retina of the eye. It has been shown that hPrp31 plays a key role in U4/U6.U5 tri snRNP formation, which is assumed to take place in Cajal bodies (CBs), subnuclear organelles of animal and plant cells. Nevertheless, it is so far unknown whether the mutations in protein hPrp31 affect the stability of the tri snRNP. Using fluorescence microscopy and biochemical methods it has been shown here how hPrp31 mutations influence the localisation of protein hPrp31 in vivo and how they influence U4/U6.U5 tri snRNP formation. |
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| Umfang: | 164 Seiten |
| DOI: | 10.17192/z2007.0821 |