Messenger RNA (mRNA) is transcribed by RNA polymerase II (Pol II) and has to undergo multiple processing events before it can be translated into a protein: a cap structure is added to its 5’ end, noncoding, intervening sequences (introns) are removed and coding exons are ligated together and a poly(A) tail is added to its 3’end. Splicing, the process of intron removal, is carried out in the spliceosome, a megacomplex comprehending up to 300 proteins. The core components of the spliceosome that directly interact with the pre-mRNA are the small nuclear ribonucleoprotein particles (snRNPs). They consist of one of the U-rich snRNAs U1, U2, U4, U5 or U6 together with several particle-specific proteins and core proteins. All mRNA processing events can occur co-transcriptionally, i.e. while the RNA is still attached to the gene via Pol II. The in vivo studies of co-transcriptional RNA processing events had been possible only in special biological systems by immunoelectron microscopy and only recently, Chromatin Immunoprecipitation (ChIP) made it possible to investigate cotranscriptional splicing factor assembly on genes. My thesis work is divided into two parts: Part I shows that the core components of the splicing machinery are recruited co-transcriptionally to mammalian genes in vivo by ChIP. The co-transcriptional splicing factor recruitment is dependent on active transcription and the presence of introns in genes. Furthermore, a new assay was developed that allows for the first time the direct monitoring of co-transcriptional splicing in human cells. The topoisomerase I inhibitor camptothecin increases splicing factor accumulation on the c-fos gene as well as co-transcriptional splicing levels, which provides direct evidence that co-transcriptional splicing events depend on the kinetics of RNA synthesis. Part II of the thesis is aimed to investigate whether Pol II has a functional role in the biogenesis of the U6 snRNA, which is the RNA part of the U6 snRNP involved in splicing. Pol III had been shown to transcribe the U6 snRNA gene, but ChIP experiments revealed that Pol II is associated with all the active U6 snRNA gene promoters. Pol II inhibition studies uncovered that U6 snRNA expression and probably 3’end formation is dependent on Pol II.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:swb:14-1157969003302-91445 |
| Date | 11 September 2006 |
| Creators | Listerman, Imke |
| Contributors | Technische Universität Dresden, Biologie, Technische Universität Dresden, Max-Planck-Institut für Molekulare Zellbiologie und Genetik, Dr. Karla Neugebauer, Prof. Dr. Francis Stewart, Prof. Dr. Neus Visa, Dr. Karla Neugebauer |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
Page generated in 0.0027 seconds