Regulation of transcript stability by splicing in non-coding gene regions

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We believe we have discovered a novel mechanism by which gene expression can be regulated. Regulation of gene expression makes one cell different from another and controls many of the ways in which cells respond to their environment. Differential regulation of gene expression, rather than differences in gene function, are also thought to underlie many of the differences between individuals. In order for a cell to express a protein from a gene it must be transcribed into RNA, which is then translated into a protein. Not all the sequence within a gene makes it into the final RNA: the "protein coding-regions" are interspersed with regions known as "introns" that must be removed by a process called splicing to reconstitute the sequence to make the protein. Regulation of gene expression is usually studied by looking at the rate of transcription, but the stability of the transcribed RNA is also important. Sequences at the end of the transcript that are not translated are called 3' Untranslated regions (3' UTRs). These UTRs contain binding sites for a range of regulatory factors (e.g. the small regulatory RNAs known as microRNAs, or RNA binding proteins such CELF1) that can alter transcript stability. Another important mechanism controlling RNA stability is called Nonsense Mediated Decay (NMD). This ensures that transcripts containing a gene-disabling mutation are destroyed. NMD also results in the destruction of transcripts that are incorrectly spliced. Splicing in the 3' UTR is thus thought to trigger NMD. Until now, RNA sequences that would have resulted from splicing in the 3' UTR have been regarded as artefacts and largely ignored. However, we have found substantial expression of thousands of such written-off RNAs, and in many cases the removed sequence contains predicted binding sites for microRNAs. This suggests two hypotheses: 1) This splicing does trigger NMD, causing a regulated destabilisation of the transcript. 2) This splicing does not trigger NMD and instead removal of UTR sequence regulates the sensitivity of RNA regulator factors such as miRNAs. We have preliminary evidence for examples of both these hypotheses. If we are correct then this will represent a novel, but fundamental mechanism of gene regulation. Here we propose to reanalyse tens of thousands of existing samples from different tissues, individuals and lab cell lines to examine the prevalence of this mechanism and how it relates to different gene expression levels between individuals and cell types. We will measure the difference in transcript stability between RNAs with splicing in the 3' UTR and those without, both in selected candidates and genome-wide. We will do this in cells with a normal NMD pathway and ones in which we have disabled the NMD pathway to determine if it is responsible for any differences. We will also ascertain whether this splicing regulates sensitivity to RNA binding factors by manipulation of the sequences of UTRs of interest and levels of the RNA binding factors. Finally we will examine the role of this mechanism in active regulation in response to pro-proliferative stimuli, such as the hormone estradiol, by measuring differences in splicing on this stimulus.