Search

Cleavage and polyadenylation are essential pre-mRNA processing reactions maturing the 3’end of almost all protein encoding eukaryotic mRNAs. Analysis of the sequences required for cleavage and polyadenylation in the human melanocortin 4 receptor (MC4R) and the human transcription factors JUNB and JUND pre-mRNAs revealed that, at least for some mammalian genes, 3’end processing of the primary transcript is independent of previously described auxiliary sequence elements located upstream or downstream of the core poly(A) sequences. The analysis of the MC4R poly(A) site, contrary to the current understanding of mammalian poly(A) sites, showed that mutations of the AUUAAA hexamer sequence had no effect on 3’end processing levels while mutations in the short DSE severely reduced cleavage efficiency. The MC4R poly(A) site uses a potent DSE and to direct maximal cleavage efficiency requires only a short upstream adenosine rich sequence. Furthermore, analysis of the endogenous A-rich human JUNB poly(A) signal validated upstream A-rich core sequences as genuine 3’end formation directing sequences in human non-canonical 3’end formation signals. The results show that a minimal human poly(A) site, similar to yeast and plants, can be defined by an adenosine rich sequence adjacent to a U/GU-rich sequence element and a cleavage site. These findings further imply that some human non-canonical poly(A) sites may be recognised via a similar DSE-dependent mechanism and may not require additional auxiliary sequence elements. Finally, results on the analysis of the EDF1 poly(A) signal show that, in a spliced environment, A-rich sequences are also 3’end formation effectors but depend on an competent upstream splicing reaction for efficient definition of the 3’end processing site.