Linkage of Nonsense Codons and RNA Splicing

University Of Texas, M.D. Anderson Cancer Center, Houston TX

Investigators

Abstract

It is widely accepted that post-transcriptional events in eukaryotic cells are compartmentalized. Transcripts are spliced by spliceosomes in the nucleus and then translocated to the cytoplasm, where ribosomes and tRNAs collaborate to read the codons in these transcripts. This view has been cast into doubt, however, by the surprising observation that premature termination (nonsense) codons (PTCs) affect not only cytoplasmic events but also nuclear-associated events. Three distinct nonsense codon-induced responses have been characterized. First, many mRNAs containing PTCs are degraded in the nuclear fraction of mammalian cells. Although there is controversy as to whether this nonsense-mediated decay (NMD) mechanism occurs in the nucleus proper, several lines of evidence implicate nuclear events as being essential to trigger it. Second, the insertion of PTCs in some genes increases the levels of alternatively spliced transcripts that skip the introduced PTC. Because RNA splicing occurs in the nucleus, this nonsense-associated altered splicing (NAS) response clearly involves the nucleus. Third, the insertion of PTCs in some genes increases the levels of their precursor mRNAs at or near the site of transcription in the nucleus. To solve the paradox of how translation signals regulate nuclear events, the molecular mechanisms responsible for NAS and NMUP will be assessed. T-cell receptor-beta (TCRbeta) transcripts will be used for this research, as the TCRbeta gene frequently acquires PTCs during normal T-cell development, and therefore mechanisms that monitor acquisition of PTCs in this gene may be critical for normal immune cell function. Objective 1 is to determine the molecular mechanism responsible for NAS, including identifying the factors essential for it to occur and determining whether it occurs by a feedback mechanism. Objective 2 is to identify the molecular mechanism responsible for NMUP, including its factor requirements and its triggering mechanism, as well as to establish whether it is an early event coupled with transcription, whether it occurs as a result of inhibited splicing or precursor mRNA stabilization, whether it depends on translation, and what regulatory elements are essential for it to occur. This research should reveal whether nonsense codon-recognition occurs in the cytoplasm, triggering a signaling mechanism that regulates nuclear events (including RNA splicing), or whether instead nonsense codons are directly read in the nucleus as part of a nuclear scanning mechanism that proofreads nascent mRNAs, a controversial notion for which there is increasing evidence.