Characterization of sequences involved in polyadenylation of Eukaryotic pre-mRNA

Abstract

The major objective of this study was to characterize the regions of poly(A) signals, which in addition to the AAUAAA are sufficient for accurate and efficient polyadenylation. In addition, this study was designed to test the effect of varying the spacing and sequence between functional elements of a poly(A) signal. Transient expression analyses in transfected COS cells, RNA mapping assays, and chloramphenicol acetyltransferase activity measurements were used to study polyadenylation of transcripts from pSV2-neo and pSV2-cat, in which the SV40 early poly(A) signal has been modified. The results presented here suggest that single site selection and efficient polyadenylation require both the AAUAAA hexamer and nucleotides located downstream from it. Sequences located downstream from the AAUAAA of adenovirus L3, mouse β-globin, SV40 early, and late transcripts can function with an exogenous hexamer, suggesting that downstream sequences are interchangeable and flexible. This flexibility is supported further by the observation that sequences not normally found in poly(A) signals can contribute to polyadenylation. The present study supports the idea that poly(A) signals are composed of at least three functional components, the AAUAAA, the cleavage site, and the downstream element. These three elements appear to be sufficient for polyadenylation with minimal or no contribution by nucleotides located upstream of the hexamer. However, these elements need to be positioned at appropriate distances from each other in order to constitute a functional poly(A) signal. This study suggests an optimal spacing of 12-22 nucleotides between the AAUAAA and the poly(A) site, and 7-13 (in SV40 late transcripts) between the cleavage site and the downstream element.