The nucleic acid data:
IRESite Id: 109 Version: 17
Originaly submitted by: Martin Mokrejš Submission date: 2005-12-24 00:00:00
Reviewed by: Martin Mokrejš Last change: 2009-09-03 14:14:10
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  nonexisting_chimera_of_GenBank_record_and_tested_IRES_fragment
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  XIAP
The genetic origin of this natural mRNA/+RNA:
  nuclear
The GenBankId GI:# number of the most similar mRNA/+RNA sequence to this one.
28290426 
The mRNA/+RNA description: 
Assembled chimeric sequence created by utilization of the 5'-UTR from GI:28290426  with the remaining sequence
from GI:32528298
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  guessed_as_the_sequence_was_never_published_by_authors_nor_described_in_sufficient_detail
The organism containing this mRNA with IRES segment in its genome:
Homo sapiens
A promoter reported in cDNA corresponding to IRES sequence:
  yes
The total number of notable open-reading frames (ORFs):
  1
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:
heterogeneous_population_of_molecules_found
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
not_tested
Integrity (uniformity) of mRNA tested using RNAi:
heterogeneous_population_of_molecules_found
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 HEK 293T/17 (ATCC CRL-11268)
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):
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:
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 HeLa (ATCC CCL-2)
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 1 Last change: 2006-08-01 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
XIAP
The description of the protein encoded in this ORF:
X-linked inhibitor of apoptosis
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  467-1960
Remarks:
For creation of this IRESite record we have used an EST fragment (which matched the described primers) merged
with Refseq mRNA sequence which had incomplete 5'-UTR. van Eden et al. (2004) actually amplified some
sequence from HeLa cells.

The tested cDNA fragment (bases 5-464 in IRESite) contains in its RNA form a splice-acceptor site as confirmed
by 2 additional bicistronic mRNA molecule types found by RT-PCR (Fig. 2). The splicing site is shown in Fig
3A. Existence of the acceptor site was also claimed to be confirmed by other EST records of XIAP mRNA
molecules. The reported splice junction is "5'-AGaAAGGUG-3'". [van Eden et al. (2004)]

In addition, the sequence between 1-434 is not supported by any dbEST data except record GI:28290426
(BX119811) supporting bases 1-484.

Use of a promoter-less plasmid to test for cryptic promoter presence did not reveal any promoter in the tested
region (but authors did not show the actual results in the article). When direct RNA transfection was
performed IRES activity was only observed in 293T cells (2-fold activity of the control empty vector pRL-FL)
while no significant activity was found in HepG2 and HeLa S3 cells. [van Eden et al. (2004)]

RNAi was used to study whether its application decreases Rluc and Fluc activity in the same scale -- which
would confirm that they originate from a physically same mRNA molecule and in turn would confirm that
1) no promoter exists in the DNA region in corresponding the the expected mRNA molecules and
2) no aberrant splicing occurs.

It was found that activity of both luciferases was NOT decreased equivalently. [van Eden et al.
(2004)]

The IRES activity was also shown in rabbit reticulocyte lysates using bicistronic capped and non-capped mRNAs.
[van Eden et al. (2004)]


Bert et al. (2006) tested XIAP IRES in HeLa cells using promoter-less plasmids with or without the enhancer
left in (Figure 2). They showed there is a cryptic promoter activatable when the enhancer is left in.
Citations:
Van Eden M. E., Byrd M. P., Sherrill K. W., Lloyd R. E. (2004) Demonstrating internal ribosome entry sites in eukaryotic mRNAs using stringent RNA test procedures. RNA. 10(4):720-730
Bert AG, Grépin R, Vadas MA, Goodall GJ (2006) Assessing IRES activity in the HIF-1alpha and other cellular 5' UTRs. RNA. 12(6):1074-1083
IRESs:
IRES:
Version: 13 Last change: 2009-09-03 14:14:10
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  XIAP_5-464
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  5-464
Conclusion:
  probably_not_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
"IRES" activity was found to overlap with the polypyrimidine tract, which unfortunately is also involved
in the splicing issues. Mutation of the polypyrimidine tract decreased dramatically by 30-fold the "IRES"
activity although still aberrantly spliced products were detected.

Similarly, mutations of the splice donor/acceptor sites resulted in lower abundance of the alternative
splice variants or even shifted the splice spliced by few bases. None of these approaches could not be used
to completely eliminate the splicing issue with pRL-XIAP-FL plasmid. [van Eden et al. (2004)]

Direct mRNA transfection confirmed only weak IRES activity in 293T cells (95% of the activity detectable
using DNA-based transfection was gone) whereas in HepG2 and HeLa cells it was not statistically significant.
The Fig. 4C,D shows that XIAP IRES seriously underperforms in contrast to HCV IRES both as a capped of
non-capped exogenous transcript. [van Eden et al. (2004)]

Several proteins have been found to interact with XIAP IRES in vitro (Lewis et al. (2007)): p37 (hnRNP A1),
p44, p45, p52 (La autoantigen), p100, p150.


Bert et al. (2006) concluded that despite the cryptic promoter there is "some" IRES activity in XIAP. Further
they report that in direct RNA transfection XIAP IRES is about 5.9x above the negative control while EMCV-R
IRES was 221x above the control (Figure 4).
Citations:
Van Eden M. E., Byrd M. P., Sherrill K. W., Lloyd R. E. (2004) Demonstrating internal ribosome entry sites in eukaryotic mRNAs using stringent RNA test procedures. RNA. 10(4):720-730
Bert AG, Grépin R, Vadas MA, Goodall GJ (2006) Assessing IRES activity in the HIF-1alpha and other cellular 5' UTRs. RNA. 12(6):1074-1083
IRES:
Version: 7 Last change: 2009-04-06 16:04:22
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  XIAP_305-466
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  305-466
Conclusion:
  probably_not_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):



Remarks:
Baranick et al., 2008 and they postulate that most of the "IRES" activity observed so far was due to the
splice artifacts (Fig. 3D).
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
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
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
IRES trans-acting factor (ITAFS):
IRES trans-acting factor (ITAF):
Version: 1 Last change: 2008-06-27 20:14:51
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
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
3.1.2. 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:
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: Martin Mokrejš Reviewed by: Martin Mokrejš
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
3.2.2. 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:
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: 2 Last change: 2009-09-02 10:02:32
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
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
Holcík M, Gordon BW, Korneluk RG (2003) The internal ribosome entry site-mediated translation of antiapoptotic protein XIAP is modulated by the heterogeneous nuclear ribonucleoproteins C1 and C2. Mol. Cell. Biol. 23(1):280-288
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: 15
Version: 2 Last change: 2009-09-03 14:14:10
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):
297-469
The underlying nucleic acid sequence and structure of the mapped region:



Remarks:
2D structure of XIAP from Figure 1. Several mutations altering in certain region the structure did not affect
the IRES activity (Figure 7).
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: 23
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]
110.00
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: 24
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]
110.00
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: 25
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]
110.00
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: 26
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]
110.00
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