The nucleic acid data:
IRESite Id: 476 Version: 3
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2008-07-12 17:29:32
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 spliced in vivo transcript containing synthetic KMI1 IRES.
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_3UTR
The name of the plasmid:
pRKMI1F
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?):
  no (not convincing)
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  no (not convincing)
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:
homogeneous_population_of_molecules_confirmed
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:
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 origin of IRES in the plasmid:
  engineered
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:
pRKMI1F.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: 1 Last change: 2008-07-12 17:29:32
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):
  192-1127
ORF
ORF position:   2
Version: 1 Last change: 2008-07-12 17:29:32
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):
  1247-2899
Remarks:
The synthetic IRES was also cloned into pBRRF promoter-less vector and no cryptic promoter was found in that
setup (Figure 1F).
Citations:
Mitchell S. A., Spriggs K. A., Bushell M., Evans J. R., Stoneley M., Le Quesne J. P., Spriggs R. V., Willis A. E. (2005) Identification of a motif that mediates polypyrimidine tract-binding protein-dependent internal ribosome entry. Genes Dev. 19(13):1556-1571
IRESs:
IRES:
Version: 2 Last change: 2008-07-12 17:29:32
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  KMI1
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1174-1231
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  47
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  104
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -73
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -16
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Synthetic IRES sequence from Figure 1 forming hairpin loop structure crucial for its activity.
Citations:
Mitchell S. A., Spriggs K. A., Bushell M., Evans J. R., Stoneley M., Le Quesne J. P., Spriggs R. V., Willis A. E. (2005) Identification of a motif that mediates polypyrimidine tract-binding protein-dependent internal ribosome entry. Genes Dev. 19(13):1556-1571
The translation experiments:
Translation results:
IRESite Translation Id: 528
Version: 1 Last change: 2008-07-12 12:01:06
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:
  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:
  10.000
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 1C. In addition, in the Figure 1E is show that RNAi knock-down of PTB-1 to 10% of the normal
level drops KMI2 IRES activity to 38%.
Citations:
Mitchell S. A., Spriggs K. A., Bushell M., Evans J. R., Stoneley M., Le Quesne J. P., Spriggs R. V., Willis A. E. (2005) Identification of a motif that mediates polypyrimidine tract-binding protein-dependent internal ribosome entry. Genes Dev. 19(13):1556-1571
Last change to the database: 2019-03-18 09:32:49 GMT+1