Piano, Fabio, West, Sean M., Polanowska, Jola, Reboul, Jerome, Gutwein, Michelle, Gunsalus, Kristin C., Mecenas, Desirea G., Bian, Wenting
[
International Worm Meeting,
2015]
Proper spatio-temporal control of gene activity is vital for C. elegans germline development and maintenance and is determined primarily by regulatory elements within 3'UTRs (Merritt et al., Curr Biol 2008). Because almost half of protein-coding genes in the genome are subject to alternative polyadenylation (Mangone et al., Science 2010; Jan et al., Nature 2011), we are investigating whether the regulatory potential of genes during germline development is controlled by alternative 3'UTR isoform expression. We have established a Low Input 3'-End Sequencing (LITE-Seq) method to simultaneously identify and quantify mRNA transcript abundance and 3'UTR isoforms from small RNA samples, and we have applied it to investigate differences in transcripts and 3'UTR isoforms expressed in oocyte- and sperm-producing germline and in three distinct developmental stages within the hermaphrodite germline (mitosis, early meiosis, and developing oocytes). We observe on a global level that 3'UTRs in sperm-producing germline tend to be shorter than those expressed in oocyte producing germline, and that 3'UTRs become progressively longer as germ cell nuclei proliferate, enter meiosis, and differentiate into oocytes. We have identified numerous transcripts whose abundance and/or 3'UTR isoforms differ in a sex- or developmental stage-dependent manner. We also detect examples of 3'UTR isoform switching between sexes or developmental stages, including for some genes whose total transcript abundance is similar. To test the idea that individual transcripts may be subject to differential post-transcriptional regulation by selective expression and/or degradation of alternative 3'UTR isoforms at different developmental times, we developed an in vivo assay that reports on the translational regulatory potential of alternative 3'UTR isoforms in the germline. The reporter construct enables the cloning of two distinct 3'UTR isoforms into a Gateway-compatible, two-color reporter system in which each fluorophore is subject to translational regulation by a single 3'UTR isoform. Using this reporter system, we found that protein expression for several genes identified above is altered in a 3'UTR isoform-dependent manner, and that protein levels vary in different developmental contexts. Future work to identify cis-regulatory elements within the variable regions of 3'UTRs will enable us to assay their relative contributions to specific spatio-temporal expression patterns of known developmental regulators in the germline and to ascertain their functional significance in different developmental processes.