The nucleic acid data:
IRESite Id: 530 Version: 5
Originaly submitted by: Václav Vopálenský
Reviewed by: Martin Mokrejš Last change: 2008-10-27 23:35:24
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
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.
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:
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.
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  no
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  not tested
A promoter reported in cDNA corresponding to IRES sequence:
  no (not convincing)
Summary of possible issues when IRES cDNA is experimentally transcribed in vivo:
Summary of experiments studying integrity of the in vivo transcripts in a particular host:
Integrity (uniformity) of mRNA tested using Northern-blot:
not_tested
Integrity (uniformity) of mRNA tested using RNase protection:
unclear_result
Integrity (uniformity) of mRNA tested using 5'-RACE:
not_tested
Integrity (uniformity) of mRNA tested using primer extension :
not_tested
Integrity (uniformity) of mRNA tested using RT-PCR:
not_tested
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
not_tested
Integrity (uniformity) of mRNA tested using RNAi:
not_tested
Integrity (uniformity) of mRNA tested using S1 nuclease mapping:
not_tested
Cryptic promoter presence was confirmed by expression from a promoter-less plasmid:
not_tested
Cryptic promoter presence was confirmed in an experimental setup involving inducible promoter:
not_tested
Integrity (uniformity) of mRNA molecules or possible promoter presence expressed in vivo was tested using another method, please specify in Remarks:
not_tested
The organism used:
Homo sapiens HEK 293T (ATCC 293tsA1609neo)
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:
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):
  189-3332
ORF
ORF position:   2
Version: 1 Last change: 2008-10-27 23:35:24
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):
  3778-4437
Remarks:
Unclear results of ribonuclease protection assay because of not good quality of Figure 2B. There are some
minor bands clearly visible on the PAA gel (Figure 2B).
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: 1 Last change: 2008-10-23 13:15:19
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):
  3622-3771
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_-150/-1 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: 578
Version: 7 Last change: 2008-10-27 23:35:24
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:
  156.100
Name of IRES used as the positive control:
  cIAP_-1222/-1
Name of the plasmid used as the positive control.
pbetaGAL/HIAP2(-1222/-1)/CAT
IRESite Id of the plasmid used as positive control.
  524
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
1565
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:
The relative IRES activity was determined in p86-cotrasfected HEK293T cells because the caspase-mediated
cleavage fragment of p97/DAP5/NAT1, p86, selectively enhances translation of c-IAP1 IRES element both in vivo
and in vitro in a manner that is independent of the translation of the upstream cistron
(Warnakulasuriyarachchi et al., 2004).
Translational data are derived from Fig. 5A.

Similar results were obtained in vitro using rabbit reticulocyte lysate, where is possible to prove the
production of CAT protein on SDS-PAGE gel, compared with the bicistronic construct with shorter the 5' UTR
insert (pbetaGAL/HIAP2(1142-1222)/CAT). See Figure 5B.
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