The functions of Mud2 and Prp19C components in transcription and TREX occupancy

Minocha, Rashmi (2018): The functions of Mud2 and Prp19C components in transcription and TREX occupancy. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Different steps in gene expression from transcription to mRNA processing and nuclear mRNP export are connected intimately for quality control. The TREX complex is a key player that couples transcription elongation to nuclear mRNA export (Strasser, Masuda et al. 2002). The Prp19 complex (Prp19C, also known as NTC complex), originally known for its role in splicing, plays a role in stabilizing TREX complex at transcribed genes. Prp19C interacts with the TREX in vivo and is also required for efficient transcription (Chanarat, Seizl et al. 2011). Furthermore, it has been reported in the mammalian cells that Prp19C is recruited to transcriptional machinery by the early splicing factor U2AF65 (David, Boyne et al. 2011). U2AF65 interacts directly with Prp19C and the phosphorylated CTD, leading to an increased recruitment of both U2AF65 and Prp19C in a CTD-dependent manner, which enhances the splicing reaction. In Saccharomyces cerevisiae, Mud2 is the potential homologue of U2AF65. In this study, a novel function of Mud2 in the gene expression, namely in transcription, was identified. Using chromatin immunoprecipitation (ChIP), it was shown that Mud2 is recruited to both intron‐containing and intronless genes in a transcription-dependent manner. Mud2 binds directly to the S2-phosphorylated CTD and S2 phosphorylation is needed for its full occupancy at transcribed genes. Furthermore, Mud2 coimmunoprecipitates Prp19C in vivo in an RNA-independent manner and is also required for maintaining Prp19C and TREX occupancy at transcribed genes. Using in vivo and in vitro transcription assays, it was shown that Mud2 is necessary for full transcriptional activity by RNA Polymerase II (RNAPII). Taken together, Mud2 can be classified as a novel transcription factor that is necessary for the recruitment of mRNA-binding proteins to transcribed genes. In addition, a direct interaction between the Prp19C and THO, a sub-complex of TREX, was shown. Prp19C consists of the essential subunits Prp19, Cef1, Clf1, Syf1, Cwc2 and Prp46 and the non-essential subunits Ntc20, Snt309, Isy1 and Syf2. However, the molecular mechanism of Prp19C function and the roles of its individual subunits in maintaining TREX at transcribed genes and in transcription have remained unknown. In this study, the three non-essential subunits of Prp19C, Isy1, Ntc20 and Syf2, were investigated for their roles in Prp19C-TREX interaction and transcription. Additionally, an NTC related protein (NTR), which is a component of a complex containing Cef1 and hence named Cwc15 (complexed with Cef1), was also analyzed. The functions of these four proteins in Prp19C and TREX occupancy as well as Prp19C-TREX, Prp19C-Mud2 and Prp19C-RNAPII interaction were determined. Taken together, this analysis shows that specific Prp19C subunits play differential roles in gene expression.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Mud2, Prp19C, TREX, transcription, mRNP formation
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 18. September 2018
• 1. Bericht­erstatter:in: Sträßer, Katja