The nucleic acid data:
IRESite Id: 215 Version: 3
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: 
Putative in vivo transcript from SV40 promoter with the chimeric intron spliced out and containing
renilla_luciferase - hairpin_from_multiple_cloning_site - Kv1.4 IRES 1.2kb - firefly_luciferase and is
terminated after the late SV40 polyA signal. An FLuc-fusion protein should be translated from the second
cistron (4 aminoacids added extra).
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_both_UTRs
The name of the plasmid:
pRSTF-Kv1.4_1.2
The name of the promoter used to express this mRNA:
  SV40
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:
unclear_result
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:
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 HeLa (ATCC CCL-2)
Summary of experiments studying integrity of the in vivo transcripts in a particular host:
Integrity (uniformity) of mRNA tested using Northern-blot:
unclear_result
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:
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:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
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):
  191-1147
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):
  2484-4148
Remarks:
Alternatively spliced mRNAs were observed in transiently transfected HeLa cells (Fig. 1D) using Northern blot.
Deletion of the chimeric intron resulted in lower FLuc activity observed (data were not shown).

Jimenez et al. (2005) in Fig. 3C have also shown that at least no strong promoter is contained in Kv1.4 but
also confirmed the aberrant splicing issue in the same figure.
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
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
IRESs:
IRES:
Version: 2 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:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1286-2483
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):


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: 187
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 (ATCC CCL-2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  189.100
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
IRESite Id of the plasmid used as positive control.
  211
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
819
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:
Data from Fig. 1B were recalculated for each organism/cell line against Fig. 1C where CVB3 positive control
was characterized (set to 100%). Beware monocistronic transcripts were observed at least in human HeLa cells,
possibly as a result of alternative splicing.
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: 188
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 CV-1 (ATCC CCL-70)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  78.100
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
IRESite Id of the plasmid used as positive control.
  211
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
819
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:
Data from Fig. 1B were recalculated for each organism/cell line against Fig. 1C where CVB3 positive control
was characterized (set to 100%). Beware monocistronic transcripts were observed at least in human HeLa cells,
possibly as a result of alternative splicing.
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: 189
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 SK-N-SH (ATCC HTB-11)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  24.800
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
IRESite Id of the plasmid used as positive control.
  211
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
819
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:
Data from Fig. 1B were recalculated for each organism/cell line against Fig. 1C where CVB3 positive control
was characterized (set to 100%). Beware monocistronic transcripts were observed at least in human HeLa cells,
possibly as a result of alternative splicing.
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: 190
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 NLF
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  10.100
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
IRESite Id of the plasmid used as positive control.
  211
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
819
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:
Data from Fig. 1B were recalculated for each organism/cell line against Fig. 1C where CVB3 positive control
was characterized (set to 100%). Beware monocistronic transcripts were observed at least in human HeLa cells,
possibly as a result of alternative splicing.
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: 191
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:
Mus musculus C2F3
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  78.600
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
IRESite Id of the plasmid used as positive control.
  211
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
819
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:
Data from Fig. 1B were recalculated for each organism/cell line against Fig. 1C where CVB3 positive control
was characterized (set to 100%). Beware monocistronic transcripts were observed at least in human HeLa cells,
possibly as a result of alternative splicing.
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: 192
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:
Rattus norvegicus PrCM
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  16.000
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
IRESite Id of the plasmid used as positive control.
  211
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
819
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:
Data from Fig. 1B were recalculated for each organism/cell line against Fig. 1C where CVB3 positive control
was characterized (set to 100%). Beware monocistronic transcripts were observed at least in human HeLa cells,
possibly as a result of alternative splicing.
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: 193
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:
  19.100
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
IRESite Id of the plasmid used as positive control.
  211
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
819
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:
Data from Fig. 1B were recalculated for each organism/cell line against Fig. 1C where CVB3 positive control
was characterized (set to 100%). Beware monocistronic transcripts were observed at least in human HeLa cells,
possibly as a result of alternative splicing.
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
Last change to the database: 2015-04-16 16:45:23 GMT+1