Poster abstracts
Poster number 113 submitted by Hamza Rahman
Purification and analysis of proteins associated with nonsense codon-containing mRNA in Saccharomyces cerevisiae
Hamza Rahman (Case Western Reserve University Depertment of genome and genomic sciences ), Dajuan Whiteside (Case Western Reserve University Depertment of genome and genomic sciences ), Kristian Baker (Case Western Reserve University Depertment of genome and genomic sciences )
Abstract:
Nonsense-mediated mRNA decay (NMD) is a highly conserved cellular RNA quality control pathway that recognizes and rapidly degrades mRNAs harboring nonsense codons. The detection and rapid elimination of these aberrant mRNAs help to promote fidelity in gene expression and prevent accumulation of truncated polypeptides that are likely non-functional or have deleterious effects in the cell. Three proteins are essential for NMD and make up the core NMD machinery - Up-frameshift 1 (Upf1), Upf2, and Upf3 – with Upf1 being the only factor with known enzymatic activity and an ability to bind directly to RNA. It remains unclear how the NMD machinery is able to discriminate between normal and aberrant mRNA and target only those harboring nonsense codons for rapid degradation. In an attempt to gain a better understanding of the mechanism of NMD substrate recognition, we have developed a protocol to isolate transcript-specific mRNA-protein complexes from normal and nonsense-containing mRNA from the model organism, Saccharomyces cerevisiae, and have identified the associated proteins by mass spectrometry. It is anticipated that differences in protein complex composition will help shed light on how nonsense-containing mRNAs are recognized by the NMD machinery.
We have identified novel proteins specifically enriched on an NMD substrate and are performing experiments to identify a potential a role for these in NMD substrate recognition. To validate and extend these initial findings, I have developing a second mRNA reporter for mRNP purification employing the endogenous PRC1 gene. I have cloned PRC1 onto a high-copy number plasmid and confirmed the construct by restriction digestion and DNA sequencing, and characterized mRNA expression by Northern blot. Northern blot results indicate that: (i) PRC1 mRNA is highly expressed from the plasmid; (ii) introduction of a nonsense codon at position 133 (i.e. PRC1PTC133) results in a dramatic reduction in the steady-state level of this reporter transcript; and, (iii) inhibition of NMD leads to an increase in the abundance of PRC1PTC133 mRNA. On-going studies with this reporter will contribute to identifying differences between NMD sensitive and insensitive mRNPs and help reveal how cells recognize and target nonsense-containing mRNA to NMD.
Keywords: NMD, RNA, Upf1