The nucleic acid data:
IRESite Id: 315 Version: 1
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2007-05-15 00:00:00
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:
  our_best_guess
The mRNA/+RNA description: 
Bicistronic in vivo transcript containing Rluc, XIAP IRES, Fluc-fusion protein. The mRNA is spliced and does
not contain the chimeric intron. The sequence ends at its 3'-end right after the poly(A) signal from bovine
growth hormone (BGH) mRNA and thus the 3'-UTR might be slightly wrong (it is unknown what region of the BGH
signal was transcribed and where the poly(A) tail was added).
The mRNA/+RNA sequence represented in the +DNA notation:


Warning: mRNA sequence when devoid of trailing 'A's is still not a substring of the plasmid sequence. Is it because an intron is spliced out? Stay calm then. :-)
Credibility of mRNA sequence:
  reverse_engineered_sequence_and_should_match_experiment_maybe_except_3UTR
The name of the plasmid:
pRL-XIAP-FL
The name of the promoter used to express this mRNA:
  CMV
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  yes
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  yes
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:
heterogeneous_population_of_molecules_found
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:
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 293T (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 HeLa (ATCC CCL-2)
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:
pRL-XIAP-FL.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: 1 Last change: 2007-05-15 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
RLuc
The description of the protein encoded in this ORF:
Renilla luciferase
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):
  194-1129
ORF
ORF position:   2
Version: 1 Last change: 2007-05-15 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FLuc
The description of the protein encoded in this ORF:
Firefly luciferase
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):
  1624-3276
Remarks:
Aberrantly spliced messages are produced from the DNA vector (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)]

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)]
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
IRESs:
IRES:
Version: 1 Last change: 2007-05-15 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  XIAP
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1145-1604
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  16
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  475
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -479
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -20
The sequence of IRES region aligned to its secondary structure (if available):


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
The translation experiments:
Translation results:
IRESite Translation Id: 401
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens 293T (ATCC CRL-1573)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  250.000
Name of IRES used as the positive control:
  HCV_type_1b
Name of the plasmid used as the positive control.
pRL-HCV-FL
Name of the plasmid used as the negative control.
pRL-FL
IRESite Id of the plasmid used as positive control.
  131
IRESite Id of the plasmid used as negative control.
  119
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  53.000
The size (length) of intercistronic region in the positive control:
485
The size (length) of intercistronic region in the negative control:
28
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 results from Fig. 1D do not form the supporting evidence for existence of IRES in XIAP (the direct mRNA
transfection is the key experiment, see IRESite Id: 314 namely its subsection IRESite Translation Id: 399 and
400). They only demonstrate how the luciferase values obtained are biased by aberrant splicing of the
transcripts (Fig. 2).
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
Translation results:
IRESite Translation Id: 402
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HeLa S3 (ATCC CCL-2.2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  5800.000
Name of IRES used as the positive control:
  HCV_type_1b
Name of the plasmid used as the positive control.
pRL-HCV-FL
Name of the plasmid used as the negative control.
pRL-FL
IRESite Id of the plasmid used as positive control.
  131
IRESite Id of the plasmid used as negative control.
  119
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  60.000
The size (length) of intercistronic region in the positive control:
485
The size (length) of intercistronic region in the negative control:
28
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 results from Fig. 1D do not form the supporting evidence for existence of IRES in XIAP (the direct mRNA
transfection is the key experiment, see IRESite Id: 314 namely its subsection IRESite Translation Id: 399 and
400). They only demonstrate how the luciferase values obtained are biased by aberrant splicing of the
transcripts (Fig. 2).
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
Translation results:
IRESite Translation Id: 403
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HepG2 (ATCC HB-8065)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  3940.000
Name of IRES used as the positive control:
  HCV_type_1b
Name of the plasmid used as the positive control.
pRL-HCV-FL
Name of the plasmid used as the negative control.
pRL-FL
IRESite Id of the plasmid used as positive control.
  131
IRESite Id of the plasmid used as negative control.
  119
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  52.000
The size (length) of intercistronic region in the positive control:
485
The size (length) of intercistronic region in the negative control:
28
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 results from Fig. 1D do not form the supporting evidence for existence of IRES in XIAP (the direct mRNA
transfection is the key experiment, see IRESite Id: 314 namely its subsection IRESite Translation Id: 399 and
400). They only demonstrate how the luciferase values obtained are biased by aberrant splicing of the
transcripts (Fig. 2).
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
Last change to the database: 2015-04-16 16:45:23 GMT+1