A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

Abstract

Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5′ element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5′ and 3′ elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3′ enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3′ splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

ACKNOWLEDGMENTS

We are grateful to Lynne Maquat and Greg Tullis for valuable advice and discussion and to Lisa Burger for excellent technical assistance.

This work was supported by PHS grant RO1 A121302 from NIAID and a grant from the Council for Tobacco Research, U.S.A., to D.J.P. A.G. was partially supported by the University of Missouri Molecular Biology Program during a portion of this work.