Length increase of the human alpha-globin 3 '-untranslated region disruptsstability of the pre-mRNA but not that of the mature mRNA

Polyadenylation increases the stability of mRNA molecules. By studying the effect of the length of 3'-untranslated region (UTR) on mRNA levels, we hav e found that alpha-globin pre-mRNA is stabilized by a mechanism that does n ot modulate the half-life of mature mRNA. The insertion of DNA fragments of various unrelated sequences into the 3'-UTR of the human alpha-globin gene strongly reduces mRNA abundance upon transfection into choriocarcinoma JEG -3 cells. We found an inverse relationship between mRNA levels and the leng th of the introduced fragments. In fact, mRNA levels as low as 1% were obse rved after inserting a 477-nucleotide (nt) fragment, whereas inserting a fr agment of 86 nt at the same position had no effect on mRNA accumulation. DN A insertion induced no change in transcription rate or in half-life of matu re mRNA. Semi-quantitative reverse transcription-polymerase chain reaction revealed that inserting a 477-nt fragment in the 3'-UTR resulted in decreas ed levels of nuclear pre-mRNA in proportion to that observed for mature mRN A. In contrast, the insertion of the 477-nt exogenous DNA in the last intro n had no effect on mRNA levels despite the presence of intronic sequences i n the pre-mRNA. This shows that the reduction of pre-mRNA level was not due to the insertion of putative ribonuclease cleavage sites or the insertion of a segment DNA that reduces the elongation efficiency. Taken together, ou r results strongly support the existence of a pre-mRNA stabilizing mechanis m that can be disrupted by increasing the length of the 3'-UTR. The fact th at the half-life of mature mRNA is not affected by DNA insertion is compati ble with a pre-mRNA-specific stabilizing mechanism that acts specifically b efore polyadenylation.