The nucleic acid data:
IRESite Id: 458 Version: 2
Originaly submitted by: Václav Vopálenský
Reviewed by: Martin Mokrejš Last change: 2008-07-08 17:05:47
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:
  3UTR_possibly_incomplete
The mRNA/+RNA description: 
Putative spliced in vivo SV40 promoter-derived transcript produced from bicistronic plasmid pRL which
comprises renilla and firefly luciferases as the first and the second cistron, respectively and the 5' UTR (nt
8 to nt 300) plus first 27 bp of CDS from human n-myc mRNA with a hairpin in front of the first cistron.
The mRNA sequence shown in this record ends at its 3'-end right after the poly(A) signal from SV40 mRNA and
thus the 3'-UTR might be slightly wrong.
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:
phpRNF
The name of the promoter used to express this mRNA:
  SV40
Description of the plasmid (facultative for promoter-less plasmid records):
Plasmid containing 5' UTR (nt 8 to nt 300) plus first 27 bp from CDS from human n-myc inserted between renilla and firefly luciferase 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:
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:
n-myc
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Homo sapiens SH-SY5Y (ATCC CRL-2266)
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:
phpRNF.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: 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):
  216-1181
ORF
ORF position:   2
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FFLuc
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):
  1527-3179
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
IRESs:
IRES:
Version: 1 Last change: 2008-07-07 02:39:56
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The IRES name:
  n-myc
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1234-1526
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  53
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  345
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -293
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:
n-myc 5' UTR (293 nt) and part of the protein coding region (27 nt)
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
The translation experiments:
Translation results:
IRESite Translation Id: 500
Version: 2 Last change: 2008-07-08 17:53:57
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Mus musculus Neuro-2a [N2a] (ATCC CCL-131)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  605.000
Name of IRES used as the positive control:
  n-myc
Name of the plasmid used as the positive control.
pRNF
IRESite Id of the plasmid used as positive control.
  72
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
345
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 positive control was used Neuro-2a [N2a] cell line transformed by similar vector pRNF lacking the
stem-loop in front of the first cistron. The hairpin loop impairs translation (by 75%) from the first
cistron while translation from the second cistron is not affected. Thus, the relative ratio 2nd/1st citron
increases.
Translational data are derived from Fig. 2B.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Last change to the database: 2019-03-18 09:32:49 GMT+1