Cis-acting elements in 3' untranslated regions (UTRs) of mRNAs are crucial to the regulation of gene expression. Animal microRNAs (miRNAs) each target hundreds of mRNAs, which are recognized by pairing to nucleotides 2-7 of the miRNA. MicroRNAs mature through sequential RNase III cleavage of characteristic stem-loop precursors. Cleavage by Drosha defines the premiRNA hairpin, which is then cleaved by Dicer to generate a mature miRNA. This biogenesis pathway ensures high fidelity definition of miRNA 5' ends, which determine target specificity. Small RNAs from Caenorhabditis elegans and Drosophila melanogaster are extensively surveyed here using high-throughput sequencing. Analysis of these libraries led to the discovery of a novel miRNA biogenesis pathway, the mirtron pathway. Unlike canonical miRNAs, mirtrons are defined by intron splicing. The excised intron lariat is debranched and folds into a pre-miRNA hairpin that is cleaved by Dicer. Because of the accuracy of the spliceosome, the mirtron pathway also allows for high fidelity miRNA maturation. The trans-acting siRNAs (tasiRNAs) found in plants also reproducibly generate discrete small RNA species. TasiRNAs align to their parent locus (a TAS gene) in a distinctive 21-nt phase. This phasing is crucial; only siRNAs in the appropriate phase have sufficient complementarity to recognize their targets. The register of this phase is established by miRNA cleavage of the TAS transcript. Analysis of siRNAs sequenced from Physcomitrella patens reveals a conserved pathway in which P. patens TAS genes all possess two cleavage sites for miR390, the miRNA that cleaves TAS3 in Arabidopsis. A second miR390 site was found in Arabidopsis TAS3 that is bound by the miRNA but not cleaved. This interaction is important in triggering tasiRNA production from TAS3 transcripts. A novel approach to mRNA 3' end identification is applied here to determine 3' UTRs in C. elegans. C. elegans UTRs are typically 150 nt long and have a higher density of miRNA seed sites than mammals. Ten percent of genes are alternatively polyadenylated. Approximately 1000 convergent gene pairs were found to use bidirectional poly(A) sites. This architecture maximizes gene density and demonstrates the influence of 3' end formation on the evolution of gene topology.