The nucleic acid data:
IRESite Id: 214 Version: 4
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2007-09-06 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:
  hopefully_full-length_mRNA
The mRNA/+RNA description: 
In vitro T7 transcript containing renilla_luciferase - hairpin_from_multiple_cloning_site -
Kv1.4 IRES - firefly_luciferase and terminated at XbaI site immediately after the STOP codon. An FLuc-fusion
protein should be translated from the second cistron (4 aminoacids added extra).
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the plasmid:
pRSTF-Kv1.4_1.2
The name of the promoter used to express this mRNA:
  T7
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  not tested
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  not tested
A promoter reported in cDNA corresponding to IRES sequence:
  not tested
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
Kv1.4
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Mus musculus
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:
pRSTF-Kv1.4_1.2.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: 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):
  12-968
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FLuc-fusion
The description of the protein encoded in this ORF:
Firefly luciferase protein with additional 4 aminoacid residues at its N-terminus
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):
  2305-3969
Citations:
Jang G. M., Leong L. E., Hoang L. T., Wang P. H., Gutman G. A., Semler B. L. (2004) Structurally distinct elements mediate internal ribosome entry within the 5'-noncoding region of a voltage-gated potassium channel mRNA. J. Biol. Chem. 279(46):47419-47430
IRESs:
IRES:
Version: 3 Last change: 2007-01-23 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  Kv1.4_1.2
The functional status of IRES:
  defective
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1107-2304
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  139
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  1336
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -1198
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -1
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
The results of Jang et al (2004) and of Jimenez et al (2005) disproved the former report of IRES
activity in KCNA4/Kv1.4 1.2, 1.0 and 0.2 kb segments by Negulescu et al. (1998).
Citations:
Jang G. M., Leong L. E., Hoang L. T., Wang P. H., Gutman G. A., Semler B. L. (2004) Structurally distinct elements mediate internal ribosome entry within the 5'-noncoding region of a voltage-gated potassium channel mRNA. J. Biol. Chem. 279(46):47419-47430
The translation experiments:
Translation results:
IRESite Translation Id: 194
Version: 2 Last change: 2007-09-09 00:00:00
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 (ATCC CCL-2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  9.100
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.500
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
133
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_without_cap_without_polyA_tail
Remarks:
The data are from Fig. 2B. Although Jang et al. (2004) did not state it explicitly the in vitro
transcripts used in the direct RNA transfection were most probably uncapped (this artificially increases the
yields from the second cistron and thus makes the IRES appear stronger and also increases the risk of degraded
RNA). In comparison with CVB3 IRES the Kv1.4 1.2kb fragment seriously underperforms. The transcripts should
have also been capped in yet another experiment. In the legend they mentioned measuring firefly
luciferase/beta-galactosidase measurements in these cells but that does not make sense as beta-galactosidase
was only measured in experiment shown in Fig. 2A.
Citations:
Jang G. M., Leong L. E., Hoang L. T., Wang P. H., Gutman G. A., Semler B. L. (2004) Structurally distinct elements mediate internal ribosome entry within the 5'-noncoding region of a voltage-gated potassium channel mRNA. J. Biol. Chem. 279(46):47419-47430
Translation results:
IRESite Translation Id: 200
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:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  1.440
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.210
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
131
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_without_cap_with_polyA_tail
Remarks:
Fig. 4A
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
Translation results:
IRESite Translation Id: 201
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:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  2.270
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.390
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
131
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_without_cap_without_polyA_tail
Remarks:
Fig. 4B, measured values provided by J. Jimenez.
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
Translation results:
IRESite Translation Id: 202
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:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  2.600
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  6.060
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
131
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_with_GpppG_cap_without_polyA_tail
Remarks:
Fig. 4B, measured values provided by J. Jimenez.
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
Last change to the database: 2015-04-16 16:45:23 GMT+1