An RNA Surveillance Mechanism that Detects Aberrant Transcripts

Miles Wilkinson, Ph.D.
Professor
Immunology Department, Box 180
U. T. M. D. Anderson Cancer Center
1515 Holcombe Blvd.
Houston, TX 77030
telephone: (713) 794-5526
fax: (713) 745-0846
email: mwilkins@mdanderson.org
prestype: Platform
presenter:= Miles Wilkinson

Jun Wang, John I. Hamilton, Mark Carter, Shulin Li, and Miles F. Wilkinson
Department of Immunology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, U.S.A.

Nonsense codons terminating translation prematurely generate potentially deleterious truncated proteins. The hazards of such dominant-negative proteins are diminished by nonsense-mediated decay (NMD), an RNA surveillance pathway that recognizes and degrades transcripts harboring premature termination codons (PTCs). Here we report that PTCs in a T-cell receptor (TCR) gene not only decrease the levels of the normally spliced (norm) mRNA but also surprisingly increase the levels of an alternatively spliced (alt) transcript that has skipped the offending PTC. RNA half-life analysis demonstrated that the increase in the amount of alt mRNA did not result from an increase in its stability. Instead, it resulted from increased splicing from the alternative splice sites used to generate the alt mRNA, as demonstrated by analyzing the levels of alt and norm spliced introns in vivo. One model that has been proposed to explain how nonsense mutations could influence RNA splicing is that such mutations disrupt splicing enhancers. We demonstrated that this splicing-enhancer disruption model does not apply in our case, as the alt TCR transcript was specifically induced by PTCs generated by point mutations and frameshifts at various positions in the rearranged VDJ exon but was not induced by missense or silent mutations. Thus, our results indicate that bona fide nonsense codons stimulate nuclear RNA splicing, which is paradoxical, as the only known machinery that recognizes nonsense codons is the translation machinery operating in the cytoplasm. We therefore examined whether alt mRNA induction in response to PTCs occurs in the nucleus or cytoplasm and whether it depends on a translation-like mechanism. Our results showed that alt mRNA induction occurred in the nuclear fraction of mammalian cells and that it had the following features of translation: (1) it depended on an inititator AUG, (2) it depended on a Kozak consensus sequence surrounding the AUG codon, (3) it was inhibited by a stem loop and suppressor tRNAs. We therefore propose that the induction of alt mRNA in response to PTCs results from a tRNA- and AUG-dependent scanning mechanism that acts in the nuclear fraction of cells.