Poster abstracts
Poster number 41 submitted by Caleb Gooden
Alternative polyadenylation of transcripts encoding trans-acting siRNAs in red clover
Caleb Gooden (Department of Plant and Soil Sciences, University of Kentucky), Randy D. Dinkins (USDA-ARS-FAPRU), Arthur G. Hunt (Department of Plant and Soil Sciences, University of Kentucky)
Abstract:
Root nodulation is a process in which host roots are infected with a soil bacterium and form nodules that better fix nitrogen from the atmosphere. In agriculture, nodules help to reduce the need for fertilizer and are thus part of sustainable practice. Studies in Medicago truncatula [1] have shown the process of root nodulation to be suppressed by the expression of a regulatory long non-coding RNA (TAS3). Over-expression of TAS3 and the miR390 pathway with which it interacts resulted in prioritization of lateral root formation over nodulation. Thus, TAS3 is an attractive target for manipulation to improve nitrogen fixation in legumes and enhance their agronomic value.
In Arabidopsis, TAS3a transcripts may end at one of two alternative polyadenylation sites, the choice of which determines whether functional or non-functional TAS3a RNAs are made [2]. Specifically, RNAs ending at the promoter-proximal site will lack the characteristic miR390 target site and thus be non-functional. TAS3a transcript ending at the distal site will possess the miR390 target site, and thus can serve as substrates for miR390-directed cleavage and subsequent production of siRNAs that regulate the responses to auxin.
We hypothesized that if two polyadenylation (poly(A)) sites are present in the TAS3 region of Trifolium pratense (red clover), then differential use of these sites would be seen in lateral roots and nodules. To test whether red clover TAS3 transcripts end at different poly(A) sites, we utilized 3’-RACE to identify and sequence TAS3 RNA isoforms. The results confirm that transcripts encoding the red clover TAS3 may end at one of two poly(A) sites. Further bioinformatics analysis showed that a miR390 target sequence is situated between the two poly(A) sites. These results confirm that red clover TAS3 transcripts undergo alternative polyadenylation much as is seen in Arabidopsis and M. truncatula, and that alternative polyadenylation may regulate TAS3 function in red clover. Current research focuses on measuring the differential use of these sites in roots and nodules using quantitative PCR (qPCR) and 3’-RACE assays. Using these results as a guide, we plan on developing transgenic approaches to manipulate the relative usages of these sites to further establish their importance on nodulation.
References:
1. Hobecker KV, Reynoso MA, Bustos-Sanmamed P, Wen J, Mysore KS, Crespi M, et al. The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth. Plant Physiol. 2017;174(4):2469-86.
2. Hunt AG. RNA regulatory elements and polyadenylation in plants. Front Plant Sci. 2011;2:109.
Keywords: alternative polyadenylation, siRNA, nodulation