The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.

Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA, Upf3 and EJC are displaced during translation. However, if there is a premature stop codon, Upf3 remains bound to EJC and marks the mutant mRNA for degradation.

Nonsense codon sequences may naturally occur in the intronic regions of an mRNA. However, a mutation can also produce a nonsense codon within a gene sequence. Such mutations are called nonsense mutations. As in the NMD pathway, these mutations also lead to premature termination of translation. The incomplete polypeptide synthesized is usually inactive. Normal function can be restored to the gene if a second mutation corrects the termination codon to an amino acid coding sequence, or suppresses the effects of the termination codon. These rectifying mutations are called nonsense suppressors. The most common nonsense suppressors are mutations in tRNA genes that produce specialized tRNAs called suppressor tRNAs. These can bind to the premature termination codon and insert an amino acid at that position.