The nucleic acid data:
IRESite Id: 486 Version: 2
Originaly submitted by: Václav Vopálenský
Reviewed by: Václav Vopálenský Last change: 2008-07-16 23:25:42
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/5'(-162)/CAT which
comprises beta galactosidase and chloramphenicol acetyl transferase as the first and the second cistron,
respectively and the part of human XIAP mRNA (from nt -162 to nt -1 of the original sequence) mRNA.
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_maybe_except_3UTR
The name of the plasmid:
pbetaGAL/5'(-162)/CAT
The name of the promoter used to express this mRNA:
  CMV
Description of the plasmid (facultative for promoter-less plasmid records):
Part of the 5' UTR from human XIAP mRNA (from nt -162 to nt -1 of the original sequence) has been cloned between the beta galactosidase and chloramphenicol acetyl transferase 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:
  no
A promoter reported in cDNA corresponding to IRES sequence:
  no
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:
not_tested
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):
homogeneous_population_of_molecules_confirmed
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:
no_promoter_confirmed
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 HEK293 (ATCC CRL-1573)
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
XIAP
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Homo sapiens
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/5'(-162)/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: Václav Vopálenský
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: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The abbreviated name of this ORF/gene:
CAT
The description of the protein encoded in this ORF:
chloramphenicol acetyl transferase
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):
  4739-5398
Citations:
Holcik M., Lefebvre C., Yeh C., Chow T., Korneluk R. G. (1999) A new internal-ribosome-entry-site motif potentiates XIAP-mediated cytoprotection. Nat. Cell. Biol. 1(3):190-192
Baird S. D., Lewis S. M., Turcotte M., Holcik M. (2007) A search for structurally similar cellular internal ribosome entry sites. Nucleic. Acids. Res. 35(14):4664-4677
IRESs:
IRES:
Version: 2 Last change: 2008-07-14 13:30:34
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The IRES name:
  XIAP
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  3621-3782
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  289
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  450
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -1118
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -957
The sequence of IRES region aligned to its secondary structure (if available):



Remarks:
XIAP IRES represents part of XIAP 5' UTR (nt from -162 to nt -1 of the original sequence). This region
retained full IRES activity comparable with the whole 5' UTR (from nt -1007 to nt -1) of XIAP mRNA.
Citations:
Holcik M., Lefebvre C., Yeh C., Chow T., Korneluk R. G. (1999) A new internal-ribosome-entry-site motif potentiates XIAP-mediated cytoprotection. Nat. Cell. Biol. 1(3):190-192
The translation experiments:
Translation results:
IRESite Translation Id: 537
Version: 2 Last change: 2008-07-13 10:56:51
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HeLa (ATCC CCL-2)
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.
pbetaGAL/CAT
IRESite Id of the plasmid used as negative control.
  237
The relative translation efficiency in % of the negative control:
  1.000
The size (length) of intercistronic region in the negative control:
336
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 data are derived from Fig. 1B.
Citations:
Holcik M., Lefebvre C., Yeh C., Chow T., Korneluk R. G. (1999) A new internal-ribosome-entry-site motif potentiates XIAP-mediated cytoprotection. Nat. Cell. Biol. 1(3):190-192
Translation results:
IRESite Translation Id: 538
Version: 2 Last change: 2009-09-03 19:21:43
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HEK293 (ATCC CRL-1573)
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.
pBiCmod
IRESite Id of the plasmid used as negative control.
  636
The relative translation efficiency in % of the negative control:
  0.300
The size (length) of intercistronic region in the negative control:
294
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 data are derived from Fig. 4A.
Citations:
Baird S. D., Lewis S. M., Turcotte M., Holcik M. (2007) A search for structurally similar cellular internal ribosome entry sites. Nucleic. Acids. Res. 35(14):4664-4677
IRES trans-acting factor (ITAFS):
IRES trans-acting factor (ITAF):
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
Type of the interaction between ITAF and the RNA subject to translation:
direct_interaction_with_rna
ITAF protein characteristics:
Version: 2 Last change: 2009-08-29 12:19:15
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
ITAF abbreviated name:
La
ITAF fullname:
La autoantigen
ITAF description (long):
La autoantigen (p52), 52 kDa RNA binding protein, predominantly localized to nucleus, unwinds the dsRNA in ATP-dependent manner, forms a dimer
3.1.2. Organisms or in vitro systems where this ITAF was functionally studied:
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
stimulatory
Method used to demonstrate ITAF effect:
in_vitro
In vitro system used to demonstrate ITAF effect:
other
Remarks:
HEK 293T cell extracts were used to demonstrate in vitro binding to XIAP RNA. Data from Figure 1A.
Citations:
Lewis S. M., Veyrier A., Hosszu Ungureanu N., Bonnal S., Vagner S., Holcik M. (2007) Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation. Mol. Biol. Cell. 18(4):1302-1311
IRES trans-acting factor (ITAF):
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
Type of the interaction between ITAF and the RNA subject to translation:
direct_interaction_with_rna
OPTIONAL: The interacting RNA base range (if any):
405-432
ITAF protein characteristics:
Version: 1 Last change: 2008-06-27 20:14:51
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
ITAF abbreviated name:
hnRNP_A1
ITAF fullname:
heterogeneous nuclear ribonucleoprotein A1
ITAF description (long):
heterogeneous nuclear ribonucleoprotein A1 (p37), 37 kDa
3.2.2. Organisms or in vitro systems where this ITAF was functionally studied:
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
inhibitory
Method used to demonstrate ITAF effect:
both
In vitro system used to demonstrate ITAF effect:
other
The organism where action of this ITAF was studied:
Homo sapiens HEK 293T/17 (ATCC CRL-11268)
Remarks:
HEK 293T cell extracts were used to demonstrate in vitro binding to XIAP RNA core RNP-binding sequence. Data
from Figures 1A and 2D.
Citations:
Lewis S. M., Veyrier A., Hosszu Ungureanu N., Bonnal S., Vagner S., Holcik M. (2007) Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation. Mol. Biol. Cell. 18(4):1302-1311
IRES trans-acting factor (ITAF):
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
Type of the interaction between ITAF and the RNA subject to translation:
direct_interaction_with_rna
OPTIONAL: The interacting RNA base range (if any):
332-353
ITAF protein characteristics:
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
ITAF abbreviated name:
hnRNP_C1/C2
ITAF fullname:
heterogeneous nuclear ribonucleoprotein C1/C2
ITAF description (long):
heterogeneous nuclear ribonucleoprotein C1/C2 (p44), 44 kDa
3.3.2. Organisms or in vitro systems where this ITAF was functionally studied:
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
required_but_available_internally
Method used to demonstrate ITAF effect:
in_vitro
In vitro system used to demonstrate ITAF effect:
other
Remarks:
HEK 293T cell extracts were used to demonstrate in vitro binding to XIAP RNA. Data from Lewis et al. (2007)
Fig. 1A and Lewis et al. (2005), Fig. 2.
Citations:
Lewis S. M., Veyrier A., Hosszu Ungureanu N., Bonnal S., Vagner S., Holcik M. (2007) Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation. Mol. Biol. Cell. 18(4):1302-1311
Lewis S. M., Holcik M. (2005) IRES in distress: translational regulation of the inhibitor of apoptosis proteins XIAP and HIAP2 during cell stress. Cell. Death. Differ. 12(6):547-553
IRES trans-acting factor (ITAF):
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
Type of the interaction between ITAF and the RNA subject to translation:
direct_interaction_with_rna
ITAF protein characteristics:
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Václav Vopálenský
ITAF abbreviated name:
PTB-1
ITAF fullname:
polypyrimidine tract-binding protein isoform 1
ITAF description (long):
polypyrimidine tract-binding protein isoform 1 binds dsRNA with (CCU)n motif where n is at least 3. By SELEX approach it was found it binds to 5'-CAGCCUGGUGCCUCUCUUUCGG-3' (Singh et al. (1995) Science 268:1173-1176) but also UCUU or UCUUC within pyrimidine-rich sequence (Perez et al. (1997) RNA3:1334-1347).
3.4.2. Organisms or in vitro systems where this ITAF was functionally studied:
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
required_but_available_internally
Method used to demonstrate ITAF effect:
both
In vitro system used to demonstrate ITAF effect:
other
The organism where action of this ITAF was studied:
Homo sapiens HEK 293T/17 (ATCC CRL-11268)
Remarks:
Deletion of the XIAP 5' UTR polypyrimidine tract causes a loss of PTB binding and a complete loss of IRES
activity, but does not disrupt the binding of other ITAFs.

Overexpression of PTB represses XIAP IRES activity.

HEK 293T cell extracts were used to demonstrate in vitro binding to XIAP RNA. Data from Figure 8A/B/C.
Citations:
Baird S. D., Lewis S. M., Turcotte M., Holcik M. (2007) A search for structurally similar cellular internal ribosome entry sites. Nucleic. Acids. Res. 35(14):4664-4677
Regions with experimentally determined secondary structures:
A region with the experimentally determined secondary structure:
IRESite 2D Struct Id: 23
Version: 1 Last change: 2008-07-14 15:28:11
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The function of the 2D structure:
IRES
The 2D structure causes frameshift:
no
The absolute position of the experimentally mapped region (the range includes START and STOP codons or their equivalents):
3621-3782
The underlying nucleic acid sequence and structure of the mapped region:



Remarks:
2D structure of XIAP from Fig 1.
4.1.1. Enzymes used to characterize at least partially the 2D structure.
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 36
The temperature (in degrees of Celsia):
22
The enzymatic method used to determine the 2D structure:
ribonuclease T1
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.00
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
100.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
120.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 37
The temperature (in degrees of Celsia):
22
The enzymatic method used to determine the 2D structure:
ribonuclease A
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.00
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
100.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
120.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 38
The temperature (in degrees of Celsia):
22
The enzymatic method used to determine the 2D structure:
ribonuclease V1
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.00
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
100.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
120.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 39
The temperature (in degrees of Celsia):
22
The enzymatic method used to determine the 2D structure:
ribonuclease T2
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.00
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
100.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
120.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Citations:
Baird S. D., Lewis S. M., Turcotte M., Holcik M. (2007) A search for structurally similar cellular internal ribosome entry sites. Nucleic. Acids. Res. 35(14):4664-4677
Last change to the database: 2015-04-16 16:45:23 GMT+1