The nucleic acid data:
IRESite Id: 260 Version: 6
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2007-11-02 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: 
In vitro T7 capped bicistronic transcript with 35b long polyA tail containing RLuc and FLuc ORFs.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  reverse_engineered_fragment_and_the_rest_is_a_guess
The name of the plasmid:
pRL-cIAP1-FL-polyA
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:
c-IAP1
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-cIAP1-FL-polyA.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):
  12-947
ORF
ORF position:   2
Version: 0
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):
  2097-3749
Remarks:
The cloned 5'-UTR by van Eden et al. (2004) region was not a complete 5'-UTR. There are 3 mutations in
the cloned sequence provided by R. Lloyd in comparison to both German and NIH cDNA projects: GI:34367137 and
GI:22382083. -297nt from initiator ATG is A instead of G, -177nt is A instead of T, -76nt is G instead of T.
As already mentioned the sequence provided to IRESite by R. Lloyd spans -1115 to +4 (while the +4 base not
shown in van Eden et al. (2004) Fig. 1A is 'G'. van Eden showed already published 5'-UTR of NM_001166.2
(GI:10880127) GenBank record which is 1159b long whereas current GI:41349435 (NM_001166.3) contains 1.4kb
untranslated region.

The intercistronic region contains incomplete 5'-UTR of c-IAP1 (-1115 to -1) followed by some 19 b as a
remnant of HCV ORF after mutation.

Integrity of T7 in vitro transcript was verified by glyoxal/DMSO-agarose gel electrophoresis after previous
column purification and DNAse treatment followed by phenol:chloroform (1:1) extraction. Integrity of RNA 8 hrs
after direct RNA transfection was confirmed by RT-PCR in human HEK 293T/17 cells (Fig. 5D).
Citations:
Van Eden M. E., Byrd M. P., Sherrill K. W., Lloyd R. E. (2004) Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA. 10(3):469-481
IRESs:
IRES:
Version: 1 Last change: 2008-10-23 16:25:42
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  c-IAP1_-1178/-64
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  963-2077
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:
  1130
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -1134
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) Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA. 10(3):469-481
The translation experiments:
Translation results:
IRESite Translation Id: 328
Version: 1 Last change: 2007-02-15 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 HepG2 (ATCC HB-8065)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  60.900
Name of IRES used as the positive control:
  HCV_type_1b
Name of the plasmid used as the positive control.
pRL-HCV-FL-polyA
Name of the plasmid used as the negative control.
pRL-FL-polyA
IRESite Id of the plasmid used as positive control.
  112
IRESite Id of the plasmid used as negative control.
  133
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  16.100
The size (length) of intercistronic region in the positive control:
419
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:
  exogenous_RNA_with_GpppG_cap_with_polyA_tail
Remarks:
The more appropriate negative control pRL-null-FL-polyA said to contain 590nt spacer derived from human PKR.
We conclude part of the protein coding region was cloned, unfortunately it is not clear which exactly. For
example, GenBank GI:4506102 has 1654b long ORF. This negative control had activity of 25% of the positive
control.

The best negative control was pRL-revHCV-FL-polyA (IRESite ID:130) with 0% activity of the positive control.

See Fig. 5C.
Citations:
Van Eden M. E., Byrd M. P., Sherrill K. W., Lloyd R. E. (2004) Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA. 10(3):469-481
Translation results:
IRESite Translation Id: 329
Version: 1 Last change: 2007-02-15 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 HEK 293T/17 (ATCC CRL-11268)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  88.100
Name of IRES used as the positive control:
  HCV_type_1b
Name of the plasmid used as the positive control.
pRL-HCV-FL-polyA
Name of the plasmid used as the negative control.
pRL-FL-polyA
IRESite Id of the plasmid used as positive control.
  112
IRESite Id of the plasmid used as negative control.
  133
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  22.500
The size (length) of intercistronic region in the positive control:
419
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:
  exogenous_RNA_with_GpppG_cap_with_polyA_tail
Remarks:
The more appropriate negative control pRL-null-FL-polyA said to contain 590nt spacer derived from human PKR.
We conclude part of the protein coding region was cloned, unfortunately it is not clear which exactly. For
example, GenBank GI:4506102 has 1654b long ORF. This negative control had activity of 54.4% of the positive
control.

The best negative control was pRL-revHCV-FL-polyA (IRESite ID:130) with 0% activity of the positive control.

See Fig. 5C.
Citations:
Van Eden M. E., Byrd M. P., Sherrill K. W., Lloyd R. E. (2004) Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA. 10(3):469-481
Translation results:
IRESite Translation Id: 330
Version: 3 Last change: 2007-10-23 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 S3 (ATCC CCL-2.2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  65.200
Name of IRES used as the positive control:
  HCV_type_1b
Name of the plasmid used as the positive control.
pRL-HCV-FL-polyA
Name of the plasmid used as the negative control.
pRL-FL-polyA
IRESite Id of the plasmid used as positive control.
  112
IRESite Id of the plasmid used as negative control.
  133
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  17.400
The size (length) of intercistronic region in the positive control:
419
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:
  exogenous_RNA_with_GpppG_cap_with_polyA_tail
Remarks:
The more appropriate negative control pRL-null-FL-polyA is said to contain 590nt spacer derived from human
PKR. We conclude part of the protein coding region was cloned, unfortunately it is not clear which exactly.
For example, GenBank GI:4506102 has 1654b long ORF. This negative control had activity of 0% of the positive
control.

The best negative control was pRL-revHCV-FL-polyA (IRESite ID:130) with 0% activity of the positive control.

See Fig. 5C.
Citations:
Van Eden M. E., Byrd M. P., Sherrill K. W., Lloyd R. E. (2004) Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA. 10(3):469-481
Last change to the database: 2015-04-16 16:45:23 GMT+1