Poster abstracts
Poster number 21 submitted by Rabab Abu Alhasan
Characterization of a large NMD complex that accumulates upon loss of UPF3 factors in human cells
Rabab Abu Alhasan (Molecular Genetics, OSU), Savanna Schutte (Molecular Genetics, OSU), Ria Alurkar (Biochemistry, OSU), Guramrit Singh (Molecular Genetics)
Abstract:
The nonsense-mediated decay (NMD) pathway detects faulty mRNAs with premature termination codons (PTC) and targets them for rapid decay to limit production of truncated proteins. NMD also regulates the abundance of normal physiological mRNAs. During NMD, prematurely terminating ribosomes are recognized by the core NMD factors UPF1, UPF2, and UPF3. While UPF1 is the main sensor and activator, the function of UPF3 in these processes is poorly understood. Our recent work suggests that human UPF3 paralogs, UPF3A and UPF3B, are non-essential but important enhancers of NMD that can function independent of their association with the exon junction complex (EJC). These findings challenge the widely accepted model that the main function of UPF3 is to bridge UPF-EJC interaction during NMD and raise new questions regarding the mechanism of NMD enhancement by UPF3. By investigating NMD complexes in wild-type (WT) and UPF3-lacking human colorectal cancer cell line (HCT116) using a sucrose cushion assay, we find that loss of UPF3B causes accumulation of UPF1/2 in a large macromolecular complex. Cross-linking experiments show that these complexes also exist in WT cells but are short-lived, and loss of UPF3B causes their abnormal accumulation. Interestingly, UPF3 paralogs exhibit notable differences in their ability to resolve this complex. We also find that the loss of UPF3B’s interaction with UPF2 and not the EJC is responsible for the accumulation of the aberrant complex. Investigation into the composition of the complex has revealed that it could either contain 40S ribosomal complex or a ~1 megadalton SMG1-8-9 complex (SMG1C), which interacts with both UPF1/2 and regulates UPF1 phosphorylation and consequently NMD. Ongoing work is focused on further characterizing the composition, function and target specificity of this aberrant complex. Overall, our work illuminates a novel role of UPF3 in activating the NMD pathway and thereby in post-transcriptional gene regulation.
Keywords: NMD complexes, UPF3, UPF2