The nucleic acid data:
IRESite Id: 488 Version: 3
Originaly submitted by: Václav Vopálenský
Reviewed by: Václav Vopálenský Last change: 2008-07-15 08:53:14
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:
  hopefully_full-length_mRNA
The mRNA/+RNA description: 
Putative in vivo CMV promoter-derived transcript produced from bicistronic plasmid pBiCAQP4 which comprises
beta galactosidase and chloramphenicol acetyl transferase as the first and the second cistron, respectively
and almost the full length 5' UTR from human AQP4 mRNA (from nt -275 to nt -1 of the original sequence) and
the first 7 nts (+1 to +7) from AQP4 ORF.
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:
  end-to-end_sequence_completely_same_as_in_the_experiment
The name of the plasmid:
pBiCAQP4
The name of the promoter used to express this mRNA:
  CMV
Description of the plasmid (facultative for promoter-less plasmid records):
Almost the full length 5' UTR from human AQP4 mRNA (from nt -275 to nt -1 of the original sequence) and the first 7 nts (+1 to +7) from AQP4 ORF 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:
AQP4
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Homo sapiens HEK 293T/17 (ATCC CRL-11268)
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:
pBiCAQP4.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):
  533-3331
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):
  3918-4577
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
IRESs:
IRES:
Version: 3 Last change: 2008-07-15 09:11:54
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The IRES name:
  AQP4
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  3626-3911
How IRES boundaries were determined:
guessed
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  295
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  580
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -292
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:
AQP4 IRES represents almost the full length 5' UTR from human AQP4 mRNA (from nt -275 to nt -1 of the original
sequence) and the first 7 nts (+1 to +7) from AQP4 ORF.
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
The translation experiments:
Translation results:
IRESite Translation Id: 540
Version: 1 Last change: 2009-09-03 19:20:08
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:
  2.000
Name of IRES used as the positive control:
  XIAP
Name of the plasmid used as the positive control.
pbetaGAL/5'(-162)/CAT
Name of the plasmid used as the negative control.
pBiCmod
IRESite Id of the plasmid used as positive control.
  486
IRESite Id of the plasmid used as negative control.
  636
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.300
The size (length) of intercistronic region in the positive control:
504
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:
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 AQP4 RNA. Data from Figure 8A.
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 (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).
3.2.2. 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:
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 AQP4 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 enhances AQP4 IRES activity approximately 2-fold.

HEK 293T cell extracts were used to demonstrate in vitro binding to AQP4 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
Last change to the database: 2015-04-16 16:45:23 GMT+1