Mechanisms of 3' End Production
What are the rules used to generate alternative polyadenylation? The majority of genes with multiple 3'UTR isoforms process their 3'ends using different PAS elements. It seems that there is a correlation between the PAS sequence itself and which 3'end is used. Genes with only one 3'UTR isoform tend to use the canonical PAS hexamer AAUAAA, while genes with two or more 3'UTR isoforms use the canonical sequence mostly in the far distal element, and a non-canonical one with a one or two nucleotide variation of the canonical site in the proximal one. How does the processing machinery discriminate between multiple PAS elements in the same 3'UTR, and "know" which one to produce?
The relative abundance of the factors that form the processing machinery seems to have a role in the decision between the 'weak' proximal sites with non-canonical PAS, or the more 'stable' distal site, which commonly uses the canonical PAS. However, this dosage-specific activity of the processing machinery is not the only mechanism, as PAS-specific processing factors also have been observed.
We are examining if the alternative 3'UTR isoforms are produced by changes in abundance of specific subunits of the cleavage complex, and if there are tissue-specific accessory factors that allow the production of specific 3'UTR isoforms. We will address the first hypothesis by quantifying the relative abundance of the cleavage complex subunits in different tissues using qPCR. To address the second hypothesis, we are preparing sensor worm strains that sense the occurrence of 3'end cleavage of a variant or PAS-less 3'UTR and report it using mCherry visualization in a tissue-specific manner (see Figure 3). The test 3'UTR isoform containing the variant element or PAS-less 3'UTR is fused to mCherry. When recombined in worms, the GFP will visualize the promoter activity (i.e. the presence of the mRNA), and the mCherry will report the occurred cleavage and polyadenylation of the tested 3'UTR. The silencing of any component of the
cleavage machinery will result in the loss of mCherry expression, because
the RNA is not polyadenylated and hence will be degraded.
We are preparing several different sensor strains corresponding to selected common variations of the canonical PAS element and representatives of the PAS-less 3'UTR. These strains express the 3'UTR in the corresponding tissue and are used in genome-wide RNA interference screens to identify genes involved in processing these variant and PAS-less elements.
Worms are particularly suitable for large-scale RNAi screens because the double-stranded RNA response can be easily induced by feeding available RNAi libraries. For each sensor strain we perform ~20,000 RNAi experiments, silencing every worm protein-coding gene and test for the loss of mCherry expression. Positive hits are re-screened, and genes that pass the second screen are studied in detail.