A large-scale analysis of human polyadenylation signals was carried out in 
silico. The most canonical AAUAAA hexamer and its 11 single-nucleotide variants 
that are most frequent in human genes were used to search for polyadenylation 
signals in the terminal sequences. Out of 18,277 poly(A) sites that were 
identified from 26,414 human genes, 82.5% of the sites were found to contain at 
least one of these 12 hexamers as a polyadenylation signal within 40 nucleotides 
upstream of the poly(A) site. The rest (17.5%) did not contain any of these 
hexamers, which suggests the existence of yet unknown signals. A total of 20,347 
terminal sequences in close proximity to 12 polyadenylation signals were 
collected using modified EST clustering technique to establish a large-scale 
database of polyadenylation signals. To characterize the 12 hexamers, the 
locations of polyadenylation signals that were identified as "authentic" and the 
uracil contents of the downstream region of the signal were examined. Based on 
this analysis, the 11 variants of the canonical AAUAAA were identified as 
possibly forming "functional" signals as AAUAAA. Moreover, the observed 
frequency of 41.9% for AAUAAA was significantly lower than those of other 
reports, suggesting that the non-canonical variants are more important in the 
polyadenylation process than frequently recognized. Since the poly(A) sites 
processed by those non-canonical variants have not been generally annotated in 
major gene databases, it is important to determine whether the variant hexamers 
could work as polyadenylation signals that may be responsible for generating 
heterogeneity of mRNAs by alternative polyadenylation.