The nucleic acid data:
IRESite Id: 522 Version: 4
Originaly submitted by: Václav Vopálenský
Reviewed by: Martin Mokrejš Last change: 2008-10-27 23:32:39
IRESite record type:
  plasmid_with_promoter_and_putative_IRES_translationally_characterized
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  3UTR_possibly_incomplete
The mRNA/+RNA description: 
Putative in vivo CMV promoter-derived transcript produced from bicistronic plasmid
hp-pbetaGAL/HIAP2(1073-1222)/CAT which comprises beta galactosidase and chloramphenicol acetyltransferase as
the first and the second cistron respectively and the part of human c-IAP1 5' UTR (nt from -150 to nt -1 of
the original sequence) mRNA mRNA cloned between them, with a hairpin in front of the first cistron.
The sequence ends at its 3'-end right after the poly(A) signal from BGH mRNA and thus the 3'-UTR might be
slightly wrong.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  reverse_engineered_sequence_and_should_match_experiment_except_3UTR
The name of the plasmid:
hp-pbetaGAL/HIAP2(1073-1222)/CAT
The name of the promoter used to express this mRNA:
  CMV
Description of the plasmid (facultative for promoter-less plasmid records):
Plasmid containing part of human c-IAP1 5' UTR (nt from -150 to nt -1 of the original sequence) inserted between beta galactosidase and chloramphenicol acetyltransferase reporter genes. A synthetic DNA fragment was inserted into the bicistronic constructs to introduce a stable stem loop structure 72 nt downstream from CMV transcription start site.
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  not tested
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  not tested
A promoter reported in cDNA corresponding to IRES sequence:
  not tested
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
c-IAP1
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Homo sapiens Jurkat
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
hp-pbetaGAL/HIAP2(1073-1222)/CAT.jpg
The total number of notable open-reading frames (ORFs):
  2
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
LacZ
The description of the protein encoded in this ORF:
beta galactosidase
The translational frameshift (ribosome slippage) involved:
  0
The ribosome read-through involved:
  no
The alternative forms of this protein occur by the alternative initiation of translation:
  not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  252-3395
ORF
ORF position:   2
Version: 1 Last change: 2008-10-27 23:32:39
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
CAT
The description of the protein encoded in this ORF:
chloramphenicol acetyltransferase
The translational frameshift (ribosome slippage) involved:
  0
The ribosome read-through involved:
  no
The alternative forms of this protein occur by the alternative initiation of translation:
  not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  3841-4392
Citations:
Warnakulasuriyarachchi D., Cerquozzi S., Cheung H. H., Holcik M. (2004) Translational induction of the inhibitor of apoptosis protein HIAP2 during endoplasmic reticulum stress attenuates cell death and is mediated via an inducible internal ribosome entry site element. J. Biol. Chem. 279(17):17148-17157
IRESs:
IRES:
Version: 2 Last change: 2008-10-23 13:13:42
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The IRES name:
  c-IAP1_-150/-1
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  3685-3834
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  290
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  439
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -156
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -7
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
c-IAP1 IRES represents part of the human c-IAP1 5' UTR (nt from -150 to nt -1).
Citations:
Warnakulasuriyarachchi D., Cerquozzi S., Cheung H. H., Holcik M. (2004) Translational induction of the inhibitor of apoptosis protein HIAP2 during endoplasmic reticulum stress attenuates cell death and is mediated via an inducible internal ribosome entry site element. J. Biol. Chem. 279(17):17148-17157
The translation experiments:
Translation results:
IRESite Translation Id: 572
Version: 5 Last change: 2008-10-23 13:13:42
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HEK 293T (ATCC 293tsA1609neo)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  100.000
Name of the plasmid used as the negative control.
hp-pbetaGAL/CAT
IRESite Id of the plasmid used as negative control.
  521
The relative translation efficiency in % of the negative control:
  13.100
The size (length) of intercistronic region in the negative control:
337
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
Translational efficiency was determined in thapsigargin (resuspended in DMSO)-treated cells.
DMSO-alone treated cells have about 10 times higher CAT production from hp-pbetaGAL/HIAP2/CAT plasmid compared
with empty hp-pbetaGAL/CAT vector.
Translational data are derived from Fig. 2C.

When rabbit reticulocyte lysates were primed with the hairpin-containing c-IAP1 IRES reporter construct
(hp-pbetaGAL/HIAP2(1073-1222)/CAT) and either the FLAGp97- or FLAG-p86-expressing plasmids, the addition of
p86 fragment resulted in the activation of c-IAP1 IRES (approximately 900 times higher production of CAT
protein, compared with the bicistronic construct that lacks the 5' UTR insert (hp-pbetaGAL/CAT)). See Figure
4D.
Citations:
Warnakulasuriyarachchi D., Cerquozzi S., Cheung H. H., Holcik M. (2004) Translational induction of the inhibitor of apoptosis protein HIAP2 during endoplasmic reticulum stress attenuates cell death and is mediated via an inducible internal ribosome entry site element. J. Biol. Chem. 279(17):17148-17157
Last change to the database: 2015-04-16 16:45:23 GMT+1