All polyadenylated mRNAs contain sequence of variable length between the coding region and the poly(A) tail. Little has been done to establish what role the length of the 3' untranslated region (3'UTR) plays in posttranscriptional regulation. Using firefly luciferase (luc) reporter mRNA in transiently transfected Chinese hamster ovary (CHO) cells, we observed that the addition of a poly(A) tail increased expression 97-fold when the length of the 3'UTR was 19 bases but that its stimulatory effect was only 2.3-fold when the length of the 3'UTR was increased to 156 bases. The effect of the luc 3'UTR on poly(A) tail function was orientation independent, suggesting that its length and not its primary sequence was the important factor. Increasing the length of the 3'UTR increased expression from poly(A)- mRNA but had little effect on poly(A)+ mRNA. To examine the effect of length on translational efficiency and mRNA stability, a 20-base sequence was introduced and reiterated downstream of the luc stop codon to generate a nested set of constructs in which the length of the 3'UTR increased from 4 to 104 bases. For poly(A)- reporter mRNA, translational efficiency in CHO cells increased 38-fold as the length of the 3'UTR increased from 4 to 104 bases. Increasing the length of the 3'UTR beyond 104 bases increased expression even further. Increasing the length of the 3'UTR also resulted in a 2.5-fold stabilization of the reporter mRNA. For poly(A)+ mRNA, the translational efficiency and mRNA half-life increased only marginally as the length of the 3'UTR increased from 27 to 161 bases. However, positioning the poly(A) tail only 7 bases downstream of the stop codon resulted in a 39-fold reduction in the rate of translation relative to a construct with a 27-base 3'UTR, which may be a consequence of the poly(A) tail-poly(A)-binding protein complex functioning as a steric block to translocating ribosomes as they approached the termination codon. The optimal length of the 3' noncoding region for maximal poly(A) tail-mediated stimulation of translation is approximately 27 bases. These data suggest that the length of the 3'UTR plays an important role in determining both the translational efficiency and the stability of an mRNA
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