Influence of the four leader sequences of the human insulin-like-growth-factor-2 mRNAs on the expression of reporter genes.
The human insulin-like-growth-factor-2 (IGF-2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader-2-containing and leader-4-containing mRNAs are completely polysomal, whereas mRNAs possessing leader-3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF-2 leader sequences on expression of the gene, IGF-2 leader-luciferase and leader-chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader-1, leader-2 and leader-4 were very similar, both at the level of mRNA and protein. Leader-3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328-906 of the leader sequence. The remaining 5' part of the leader sequence was efficient both in RNA expression and in translation, but the 3' part of the leader (nucleotides 906-1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA. To evaluate the effect of the IGF-2 leaders on in vitro translation, leader-chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader-1-chloramphenicol acetyltransferase mRNA translated over 20-fold less efficiently, whereas leader-2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3-5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader-3-containing mRNAs in reticulocyte lysates was barely detectable. The whole 5' region of leader-3, up to nucleotide 614, could be shown to be repressive. Only leader-4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader-1 and leader-2, this L4-chloramphenicol acetyltransferase mRNA translated in a cap-dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue. We conclude that all four IGF-2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF-2 mRNAs differentially.
De Moor
C H
CH
Department of Pediatrics, Utrecht University, The Netherlands.
Jansen
M
M
Bonte
E J
EJ
Thomas
A A
AA
Sussenbach
J S
JS
Van den Brande
J L
JL
D001483:Base Sequence; D002461:Cell Line, Transformed; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000737:chemistry*; D012314:RNA Cap Analogs / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D021382:Protein Sorting Signals; D012314:RNA Cap Analogs; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D007335:Insulin-Like Growth Factor II; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase