Identification and functional characterisation of RAM, a novel and essential component of RNA guanine-7 methylation

Gene expression in eukaryotes is dependent on the N-7 methylguanosine cap, located at the 5’ end of RNA pol II transcripts, which marks pre-mRNA for processing, stabilisation and translation initiation. The enzymes that catalyse the formation of the N-7 methylguanosine cap are recruited to RNA pol II at the initial stages of transcription. The final step in this process, N-7 methylation of the guanosine cap, is catalysed by the RNA guanine-7 methyltransferase, RNMT.<br/>RNA guanine-7 methylation is an essential process for cell viability and its up-regulation has been associated with cell transformation. However, the mechanistic details of RNMT function in mammalian cells remain elusive. In order to gain better understanding of the molecular mechanisms associated with RNA guanine-7 methylation, cellular RNMT complexes were purified from human cells and constituent proteins were identified using mass spectrometry. A novel component of the RNA guanine-7 methyltransferase complex was identified and designated as RAM (RNMT activating mini-protein). The vast majority of RNMT is found in a complex with RAM and vice versa. <br/>RAM is an RNA-binding protein, promoting recruitment of RNA to RNMT. RAM increases recombinant and cellular RNMT cap methyltransferase activity and is required for cap methylation in vivo. We therefore, describe RAM as an “obligate activator” of the human cap methyltransferase. As expected of a protein essential for cap methylation, RAM is required for gene expression, and RAM depletion results in loss of cell viability. Current studies are being focused on determining RAM/RNMT crystal structure as well as determining how the RNA guanine-7 methyltransferase complex is regulated within cells.<br/>

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2012

Author dc:creator

• Gonatopoulos-Pournatzis, Thomas

Advisor dc:contributor.advisor

• Cowling, Victoria

• Gene expression
• Cap methylation
• RNA processing

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/a3138557-b75e-4534-a5db-47f09df416e5

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/a3138557-b75e-4534-a5db-47f09df416e5