A large-scale analysis of mRNA polyadenylation in human and mouse genes was conducted, revealing that approximately 54% of human genes and 32% of mouse genes have alternative polyadenylation sites. These sites are conserved between human and mouse orthologs, indicating their importance in generating alternative gene transcripts. Genes with specific functional annotations show bias in polyadenylation configuration, with some genes having a single poly(A) site and others having multiple sites in different exons. Many poly(A) sites contain multiple cleavage sites, leading to heterogeneous 3' end formation for transcripts, suggesting that polyadenylation is imprecise. Different types of poly(A) sites have distinct nucleotide compositions in surrounding genomic regions. This study provides insights into the mechanisms of polyadenylation in mammals and highlights the role of alternative polyadenylation in gene expression regulation. The findings suggest that alternative polyadenylation is widely used by humans and mice to produce variable gene products, including mRNAs and proteins. The study also identified that genes encoding extracellular proteins often have single poly(A) sites, while genes encoding intracellular proteins tend to have alternative poly(A) sites. The results indicate that alternative polyadenylation is an evolutionarily conserved process and plays a significant role in mRNA stability and regulation. The study also found that the 3'-most poly(A) sites are the most frequently used, and that alternative polyadenylation is often associated with splicing events. The findings highlight the importance of polyadenylation in gene regulation and the need for further research to understand its role in various biological processes.A large-scale analysis of mRNA polyadenylation in human and mouse genes was conducted, revealing that approximately 54% of human genes and 32% of mouse genes have alternative polyadenylation sites. These sites are conserved between human and mouse orthologs, indicating their importance in generating alternative gene transcripts. Genes with specific functional annotations show bias in polyadenylation configuration, with some genes having a single poly(A) site and others having multiple sites in different exons. Many poly(A) sites contain multiple cleavage sites, leading to heterogeneous 3' end formation for transcripts, suggesting that polyadenylation is imprecise. Different types of poly(A) sites have distinct nucleotide compositions in surrounding genomic regions. This study provides insights into the mechanisms of polyadenylation in mammals and highlights the role of alternative polyadenylation in gene expression regulation. The findings suggest that alternative polyadenylation is widely used by humans and mice to produce variable gene products, including mRNAs and proteins. The study also identified that genes encoding extracellular proteins often have single poly(A) sites, while genes encoding intracellular proteins tend to have alternative poly(A) sites. The results indicate that alternative polyadenylation is an evolutionarily conserved process and plays a significant role in mRNA stability and regulation. The study also found that the 3'-most poly(A) sites are the most frequently used, and that alternative polyadenylation is often associated with splicing events. The findings highlight the importance of polyadenylation in gene regulation and the need for further research to understand its role in various biological processes.