Poster abstracts

Poster number 32 submitted by Laura de Lorenzo Barrios

Genome-wide analysis of stability & translatability in CAP-associated RNAs in Arabidopsis

Laura de Lorenzo (Department of Plant and Soil Sciences. University of Kentucky), Arthur G. Hunt (Department of Plant and Soil Sciences. University of Kentucky)

Abstract:
Genome-wide analysis of stability & translatability in CAP-associated RNAs in Arabidopsis
Laura de Lorenzo & Arthur G. Hunt
Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY, 40546-0312, USA.

Key words: Alternative polyadenylation, PAT-seq, stability and/or translation.


Posttranscriptional regulation of mRNA plays an important role in gene expression. One mode of control that is accomplished through RNA processing is the alternative polyadenylation (APA) (1). Polyadenylation can occur upstream, downstream and within open reading frames. Among them, coding region-APA (CAP) is the least understood. Such events should lead to transcripts without an in-frame stop codon, and affected mRNAs should be subject to rapid degradation via the nonstop decay mechanism. Thus, CAP-associated RNAs should be relatively unstable, and their translation products likewise short-lived. In eukaryotes, non-stop RNAs are subject to a battery of negative regulatory processes. In yeast, non-stop RNAs are rapidly degraded by the exosome, and the protein products of translation of non-stop mRNAs are degraded by the proteasome (2). In mammals, translation of non-stop RNAs is inhibited at a step after initiation but before the end of the non-stop RNA is reached (3).
In Arabidopsis, the paradoxical outcomes of polyadenylation within coding regions, raises questions as to if coding-region polyadenylation is linked with non-stop decay. To investigate this, we are conducting a study to analyze the stability and translatability of CAPs-associated RNAs using a genome-scale approach. In order to test the stability of RNA that end at CAPs in relation to their full-length counterparts, the genome-wide poly(A) profiles in samples treated with cordycepin (or with buffer, for controls) have been determined and the relative poly(A) site usage assessed on a gene-by-gene basis. In the same way, the translatability of CAP-associated RNAs have been tested by assessing the 3’ end profiles of mRNAs associated with polysomes (or total RNA, as control). To confirm the global poly(A) result, RT/PCR of candidates genes were carried out. Our results show that CAP-associated RNAs are stable and are found associated with polysomes (as in other eukaryotes). Future analyses will be carried out to evaluate if CAP-associated RNAs are less stable that RNAs encoded by the same gene that end at “normal” poly(A) site; and to analyze the activity and degradation of the translated protein. This will support the hypothesis that CAP utilization is a mechanism of negative regulation.

References:
1. Hunt, A.G (2014) The Arabidopsis polyadenylation factor subunit CPSF30 as conceptual link between mRNA polyadenylation and cellular signaling. Current Opinion in Plant Biology. 21: 128-132.
2. Wilson, M.A., S. Meaux, and A. van Hoof (2008). Diverse aberrancies target yeast mRNAs to cytoplasmic mRNA surveillance pathways. Biochim Biophys Acta. 1779: 550-557.
3. Akimitsu, N., J. Tanaka, and J. Pelletier (2007). Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells. EMBO Journal. 26: 2327-2338.

Keywords: Alternative polyadenylation, PAT-seq, stability and translation