Different mechanisms preserve translation of programmed cell death 8 and JunB in virus-infected endothelial cells.
OBJECTIVE
Translation initiation of eukaryotic mRNAs typically occurs by cap-dependent ribosome scanning mechanism. However, certain mRNAs are translated by ribosome assembly at internal ribosome entry sites (IRESs). Whether IRES-mediated translation occurs in stressed primary human endothelial cells (ECs) is unknown.


RESULTS
We performed microarray analysis of polyribosomal mRNA from ECs to identify IRES-containing mRNAs. Cap-dependent translation was disabled by poliovirus (PV) infection and confirmed by loss of polysome peaks, detection of eukaryotic initiation factor (eIF) 4G cleavage, and decreased protein synthesis. We found that 87.4% of mRNAs were dissociated from polysomes in virus-infected ECs. Twelve percent of mRNAs remained associated with polysomes, and 0.6% were enriched ≥2-fold in polysome fractions from infected ECs. Quantitative reverse transcription-polymerase chain reaction confirmed the microarray findings for 31 selected mRNAs. We found that enriched polysome associations of programmed cell death 8 (PDCD8) and JunB mRNA resulted in increased protein expression in PV-infected ECs. The presence of IRESs in the 5' untranslated region of PDCD8 mRNA, but not of JunB mRNA, was confirmed by dicistronic analysis.


CONCLUSIONS
We show that microarray profiling of polyribosomal mRNA transcripts from PV-infected ECs successfully identifies mRNAs whose translation is preserved in the face of stress-induced, near complete cessation of cap-dependent initiation. Nevertheless, internal ribosome entry is not the only mechanism responsible for this privileged translation.
Division of Vascular Surgery, University of Utah, Salt Lake City, USA.
Huimiao
H
Jiang
Hansjörg
H
Schwertz
Douglas I
DI
Schmid
Brandt B
BB
Jones
John
J
Kriesel
Mark L
ML
Martinez
Andrew S
AS
Weyrich
Guy A
GA
Zimmerman
Larry W
LW
Kraiss
D020121:5' Untranslated Regions; D051033:Apoptosis Inducing Factor / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism / Q000821:virology*; D020869:Gene Expression Profiling / Q000379:methods; D017930:Genes, Reporter; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism / Q000821:virology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D017955:Poliovirus / Q000472:pathogenicity*; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis* / Q000235:genetics; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism / Q000821:virology*; D014162:Transfection
D020121:5' Untranslated Regions; C494829:AIFM1 protein, human; D051033:Apoptosis Inducing Factor; D016755:Proto-Oncogene Proteins c-jun; D012315:RNA Caps; D012333:RNA, Messenger