Length Increase of the Human 
-Globin 3'-Untranslated
Region Disrupts Stability of the Pre-mRNA but Not That of the
Mature mRNA*
Pierre R.
Provost and
Yves
Tremblay
§
From the Laboratory of Ontogeny and Reproduction, Centre
Hospitalier Universitaire de Québec, Pavillon CHUL, and the
Department of Obstetrics and Gynecology, Laval
University, Québec G1V 4G2, Canada
Polyadenylation increases the stability of
mRNA molecules. By studying the effect of the length of
3'-untranslated region (UTR) on mRNA levels, we have found that
-globin pre-mRNA is stabilized by a mechanism that does not
modulate the half-life of mature mRNA. The insertion of DNA
fragments of various unrelated sequences into the 3'-UTR of the
human -globin gene strongly reduces mRNA abundance upon
transfection into choriocarcinoma JEG-3 cells. We found an inverse
relationship between mRNA levels and the length of the introduced
fragments. In fact, mRNA levels as low as 1% were observed after
inserting a 477-nucleotide (nt) fragment, whereas inserting a fragment
of 86 nt at the same position had no effect on mRNA accumulation.
DNA insertion induced no change in transcription rate or in half-life
of mature mRNA. Semi-quantitative reverse transcription-polymerase
chain reaction revealed that inserting a 477-nt fragment in the 3'-UTR
resulted in decreased levels of nuclear pre-mRNA in proportion to
that observed for mature mRNA. In contrast, the insertion of the
477-nt exogenous DNA in the last intron had no effect on mRNA
levels despite the presence of intronic sequences in 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, our results strongly support the existence of a pre-mRNA stabilizing mechanism that can be disrupted by increasing the length of the 3'-UTR.
The fact that the half-life of mature mRNA is not affected by DNA
insertion is compatible with a pre-mRNA-specific stabilizing mechanism that acts specifically before polyadenylation.
*
This work was supported by Medical Research Council of
Canada Grant MT 14365 (to Y. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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