T -cell receptor nonsense-associated altered splicing
Transcripts containing premature termination (nonsense) codons (PTCs) are harmful to cells because if translated, they can produce truncated proteins with deleterious dominant negative or gain-of-function activity. Nonsense-mediated mRNA decay (NMD) prevents the production of these proteins by selectively degrading PTC-containing messages. While often beneficial, NMD can be harmful in some cases by causing the deficiency of an essential protein. Compensating for this problem is a second response triggered by PTCs, nonsense-associated altered splicing (NAS). NAS is a mechanism that skips the PTC in the message, allowing for the translation of a protein that is truncated but occasionally functional. Evidence suggests that this putative correction mechanism can be triggered through one of two distinct ways: disruption of a splicing element such as an exonic splicing enhancer (ESE, class-I), or disruption of the open reading frame (class-II). Here, using T-cell receptor (TCR) as a model system, I demonstrate for the first time that a single type of transcript can be subject to both classes of NAS. I found mutations at sites that disrupted consensus ESEs elicited the upregulation of an alternatively spliced transcript regardless of whether or not the reading frame was disrupted, indicative of class-I NAS. In contrast, mutations at sites that did not disrupt consensus ESEs elicited upregulation of the alternatively spliced transcript only when reading frame was disrupted, indicative of class-II NAS. RNAi depletion of the essential NMD factor UPF1 showed that class-II NAS required UPF1, while class-I did not, further distinguishing between the two NAS classes. In addition, I provide more evidence that NMD and NAS are two distinct mechanisms by their different factor requirements. RNAi-mediated depletion of factors essential for NMD, such as the UPF1 kinase SMG1 and the EJC factor eIF4AIII, partially reversed NMD but showed no statistically significant effect on NAS. Knockdown of the NMD factors UPF3a or UPF3b also produced no effect on NAS. Finally, I present results suggesting that upregulation of the alternatively spliced transcript by NAS does not occur through an in trans mechanism.
Chang, Yao-Fu, "T -cell receptor nonsense-associated altered splicing" (2007). Texas Medical Center Dissertations (via ProQuest). AAI3256545.