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
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.
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.
The IRES absolute position (the range includes START and STOP codons or their equivalents): 5-464
How IRES boundaries were determined: experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):
"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).
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:
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.