A cis-acting element in the 3'-untranslated region of human TNF-alpha mRNA renders splicing dependent on the activation of protein kinase PKR.
We report a role for the 3'-untranslated region in control of mRNA splicing and show that human TNF-alpha 3' UTR harbors a cis-acting element that renders splicing of precursor transcripts dependent on activation of PKR, the RNA-activated protein kinase that phosphorylates eukaryotic initiation factor 2 (eIF2). When this element, designated 2-APRE, is present, splicing becomes sensitive to inhibition by the PKR inhibitor, 2-aminopurine, or by coexpression of transdominant-negative mutant PKR. Our results reveal that activation of PKR is required for splicing of mRNA when precursor transcripts contain the 2-APRE and that increased expression of wild-type PKR enhances their splicing efficiency. Thus, PKR responds as trans-acting factor to the 2-APRE. 2-APRE RNA forms a stable, 17-bp stem-loop structure and strongly activates PKR in vitro, inducing eIF2alpha phosphorylation. Despite its ability to activate PKR during splicing, the 2-APRE within the 3' UTR does not affect translation efficiency of the resulting TNF-alpha mRNA in transfected cells. PKR and the 3' UTR thus interact during mRNA splicing to confer a novel type of regulation on expression of the TNF-alpha gene.
Osman
F
F
Department of Molecular Virology, The Hebrew University-Hadassah Medical School, 91120 Jerusalem, Israel.
Jarrous
N
N
Ben-Asouli
Y
Y
Kaempfer
R
R
D015075:2-Aminopurine / Q000494:pharmacology; D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D019892:eIF-2 Kinase / Q000235:genetics* / Q000378:metabolism*
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D015075:2-Aminopurine; D015500:Chloramphenicol O-Acetyltransferase; D019892:eIF-2 Kinase