The nucleic acid data:
IRESite Id: 25 Version: 12
Originaly submitted by: Tomáš Mašek Submission date: 2005-06-26 00:00:00
Reviewed by: Václav Vopálenský Last change: 2009-09-04 15:04:05
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:
  both_UTRs_incomplete
The mRNA/+RNA description: 
Full-length 5'-UTR (1-395 bp) of c-myc mRNA transcribed from major P2 promoter inserted between renilla and
firefly luciferases as PvuII-NcoI insert
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:
  end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the plasmid:
pRMF
The name of the promoter used to express this mRNA:
  SV40
Aliases of the plasmid name:
Alias: pGL3Rutr
Alias: pRmycF
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 (not convincing)
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:
homogeneous_population_of_molecules_confirmed
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:
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 HeLa (ATCC CCL-2)
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
c-myc
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Homo sapiens
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


Plasmid sequence verified (partially/completely) by IRESite (more details in Remarks):
  plasmid sequence confirmed by IRESite curators by restriction analysis + parts by PCR + sequencing
GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pRMF.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: 2 Last change: 2008-05-22 18:07:23
Originaly submitted by: Tomáš Mašek Reviewed by: Václav Vopálenský
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  192-1127
ORF
ORF position:   2
Version: 3 Last change: 2008-05-22 18:07:23
Originaly submitted by: Tomáš Mašek Reviewed by: Václav Vopálenský
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1574-3226
Remarks:
Please note that the very last 2 bases of 5'-UTR were not cloned into the pGL3Rutr alias pRMF vector to keep
the putative IRES 'in frame' with the initiator ATG codon (Evans et al. (2003), Fig. 1B legend) as they
were replaced by 'cc' of NcoI site 'ccATGg'. Further, the same article describes positions of restriction
sites in Fig. 5A. Somehow, the distances of cleavage sites of PvuII and of NcoI do not match the sequence
deposited in IRESite although the pRMF sequence in the intercistronic region has been confirmed by sequencing
by IRESite curators.

It is interesting that by RNase protection Stoneley et al. (1998) show the transcripts are uniform. In
contrast, the same group in Coldwell et al. (2000) mentions that also with pRMF they observed (supposedly on
Northern-blot) possibly alternatively spliced product with size around 1.3kb in a ratio 0.8:1.0 compared to
the unspliced, full transcript. See Fig. 5 in Coldwell et al. (2000).
Citations:
Stoneley M., Paulin F. E., Le Quesne J. P., Chappell S. A., Willis A. E. (1998) C-Myc 5' untranslated region contains an internal ribosome entry segment. Oncogene. 16(3):423-428
Evans J. R., Mitchell S. A., Spriggs K. A., Ostrowski J., Bomsztyk K., Ostarek D., Willis A. E. (2003) Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo. Oncogene. 22(39):8012-8020
IRESs:
IRES:
Version: 7 Last change: 2008-07-08 19:15:06
Originaly submitted by: Tomáš Mašek Reviewed by: Václav Vopálenský
The IRES name:
  c-myc_1-395
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1178-1571
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  51
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  444
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -396
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -3
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
The entire 5'-UTR (1-395) of c-myc P2 transcript.

Stoneley et al. (2000) have shown in Figure 2 that the IRES is most active in HeLa cells, with decreasing
activity in MRC5, HepG2, GM637, HK293, COS7, MCF7, Balb/c 3T3, MEL cell lines. In the same article it was
concluded that c-myc IRES requires a non-canonical translation initiation cofactor like entero- and rhinovirus
IRESs and unlike cardio- and aphtovirus IRESs (reported by Borman et al. (1997), Nucleic Acids Res.
25:925-932).
Citations:
Stoneley M., Paulin F. E., Le Quesne J. P., Chappell S. A., Willis A. E. (1998) C-Myc 5' untranslated region contains an internal ribosome entry segment. Oncogene. 16(3):423-428
Stoneley M., Subkhankulova T., Le Quesne J. P., Coldwell M. J., Jopling C. L., Belsham G. J., Willis A. E. (2000) Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment. Nucleic Acids Res. 28(3):687-694
The translation experiments:
Translation results:
IRESite Translation Id: 41
Version: 7 Last change: 2008-05-29 16:03:37
Originaly submitted by: Tomáš Mašek Reviewed by: Václav Vopálenský
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:
  100.000
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the negative control:
  1.700
The size (length) of intercistronic region in the negative control:
67
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:
As a negative control was used HeLa cell line transformed by control, empty vector pGL3R (later named pRF).
Data from Fig. 2b. The original relative renilla/firefly luciferase activities were normalized by authors
against beta-galactosidase activity (co-transfection control).
Citations:
Stoneley M., Paulin F. E., Le Quesne J. P., Chappell S. A., Willis A. E. (1998) C-Myc 5' untranslated region contains an internal ribosome entry segment. Oncogene. 16(3):423-428
Translation results:
IRESite Translation Id: 418
Version: 1 Last change: 2008-07-09 09:02:00
Originaly submitted by: Martin Mokrejš Reviewed by: Václav Vopálenský
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:
  769.000
Name of IRES used as the positive control:
  HRV-2
Name of the plasmid used as the positive control.
pRHRVF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  467
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  10.000
The size (length) of intercistronic region in the positive control:
637
The size (length) of intercistronic region in the negative control:
67
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:
Fig. 3b
Citations:
Coldwell M. J., Mitchell S. A., Stoneley M., MacFarlane M., Willis A. E. (2000) Initiation of Apaf-1 translation by internal ribosome entry. Oncogene. 19(7):899-905
Translation results:
IRESite Translation Id: 419
Version: 1 Last change: 2008-05-29 16:03:37
Originaly submitted by: Martin Mokrejš Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  0
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as negative control.
  231
The relative translation efficiency in % of the negative control:
  0
The size (length) of intercistronic region in the negative control:
67
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_cytoplasmic_uncapped_T7_transcript_without_polyA_tail
Remarks:
The results were not shown in the article with the conclusion that a "nuclear experience" is necessary for
cellular IRESs while is not for EMCV and HRV IRESs. Similar results were obtained with exogenous bicistronic
GpppG capped poly(A30)+ run-off transcripts produced from SP6-based plasmids.
Citations:
Stoneley M., Subkhankulova T., Le Quesne J. P., Coldwell M. J., Jopling C. L., Belsham G. J., Willis A. E. (2000) Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment. Nucleic Acids Res. 28(3):687-694
Translation results:
IRESite Translation Id: 427
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Václav Vopálenský
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:
  100.000
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the negative control:
  1.400
The size (length) of intercistronic region in the negative control:
67
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:
As a negative control was used HepG2 cell line transformed by control, empty vector pGL3R (later named pRF).
Data from Fig. 2b. The original relative renilla/firefly luciferase activities were normalized by authors
against beta-galactosidase activity (co-transfection control).
Citations:
Stoneley M., Paulin F. E., Le Quesne J. P., Chappell S. A., Willis A. E. (1998) C-Myc 5' untranslated region contains an internal ribosome entry segment. Oncogene. 16(3):423-428
Translation results:
IRESite Translation Id: 516
Version: 1 Last change: 2008-07-08 18:15:26
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 NIH/3T3 (ATCC CRL-1658)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  100.000
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the negative control:
  0
The size (length) of intercistronic region in the negative control:
67
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. 5A.
Citations:
Lang K. J. D., Kappel A., Goodall G. J. (2002) Hypoxia-inducible factor-1alpha mRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell. 13(5):1792-1801
Translation results:
IRESite Translation Id: 520
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Václav Vopálenský
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:
  667.000
Name of IRES used as the positive control:
  EMCV-R
Name of the plasmid used as the positive control.
pREMCVF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  349
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  11.000
The size (length) of intercistronic region in the positive control:
619
The size (length) of intercistronic region in the negative control:
67
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 Figure 5.
Citations:
Stoneley M., Subkhankulova T., Le Quesne J. P., Coldwell M. J., Jopling C. L., Belsham G. J., Willis A. E. (2000) Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment. Nucleic Acids Res. 28(3):687-694
Translation results:
IRESite Translation Id: 525
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Václav Vopálenský
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:
  165.000
Name of IRES used as the positive control:
  EMCV-R
Name of the plasmid used as the positive control.
pREMCVF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  349
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  2.500
The size (length) of intercistronic region in the positive control:
619
The size (length) of intercistronic region in the negative control:
67
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 are from Figure 2B.
Citations:
Coldwell M. J., deSchoolmeester M. L., Fraser G. A., Pickering B. M., Packham G., Willis A. E. (2001) The p36 isoform of BAG-1 is translated by internal ribosome entry following heat shock. Oncogene. 20(30):4095-4100
Last change to the database: 2015-04-16 16:45:23 GMT+1