Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3#-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3#-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a singlenucleotide profile scan on the 3#-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which led to a prediction of the formation of secondary structures. Using an RNA secondary structure prediction program, mFold, we identified three main types of secondary structures on the sequences analyzed. Surprisingly, these observed secondary structures were all interrupted in previously constructed mutations in these regions. These results will enable us to revise the current model of plant poly(A) signals and to develop tools to predict 3#-ends for gene annotation.Messenger RNA polyadenylation is a crucial step during the maturation of most eukaryotic mRNA, in which a polyadenine [poly(A)] tract is added to the cleaved 3#-end of a precursor mRNA (pre-mRNA) posttranscriptionally. Such a modification of mRNA has been shown to affect its stability, translatability, and nuclear-to-cytoplasmic export (Zhao et al., 1999). The posttranscriptional processing of mRNA is an event that has also been found tightly coupled with splicing and transcription termination (Proudfoot et al., 2002;Proudfoot, 2004). Thus, it is an essential processing event and the integral part of gene expression.The polyadenylation process requires two major components: the cis-elements or poly(A) signals of the pre-mRNA, and the trans-acting factors that carry out the cleavage and addition of the poly(A) tail at the 3#-end. These trans-acting factors are a complex of about 25 to 30 proteins involved in signal recognition, cleavage, and polyadenylation (Proudfoot, 2004). These proteins seem to be conserved among eukaryotic organisms. However, the poly(A) signals have been found to differ widely among yeast (Saccharomyces cerevisiae), animals, and plants in terms of signal locations and sequence content. The highly conserved AAUAAA element in mammals becomes a minor signal in plant and yeast genes, and the ubiquitous downstream elements of mammalian pre-mRNAs are nowhere to be found in yeast and plants. The latter two possess an enhancing element located far upstream of the cleavage site (Zhao et al., 1999).Previous understanding of these signal elements was derived mostly...