The nucleic acid data:
IRESite Id: 586 Version: 5
Originaly submitted by: Martin Mokrejš Submission date: 2009-04-14 15:10:25
Reviewed by: Martin Mokrejš Last change: 2009-04-30 11:36:54
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 vivo spliced transcript derived from CMV IE promoter containing Renilla and firefly luciferases separated
by putative LamB1 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:
  end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the plasmid:
pR-LamB1-F
The name of the promoter used to express this mRNA:
  CMV_IE
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:
  unclear
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:
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:
unclear_result
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
unclear_result
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:
unclear_result
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:
Mus musculus MIM-R
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
LamB1
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:
pR-LamB1-F.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):
  219-1154
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):
  1602-3254
Remarks:
Based on the Figure 4B it seems that almost 20% of RLuc:FLuc activity decreased due to the LamB1 insert. That
should be related to the length of the intercistronic region: 112bp for pR-F vs. 447bp for pR-LamB1-F.

The experiments utilizing pGem-based vectors aimed to rule out presence of a cryptic promoter. The pGem
plasmid contains only T7 (in sense with FLuc) and SP6 promoters (in antisense). However, there is no
eukaryotic transcription terminator (the poly(A) signal). One can just hypothesize based on the genome
replication of SV40 and polioma viruses that concatenated transcripts encompassing (e.g. 6-8 full-length
genomic chunks would be produced) could have been detected in case there was a cryptic promoter in front of
the FLuc CDS (Petz et al. (2007), Figure 4A). The sequence of both pGem-based vector was provided directly by
M. Petz (pGem-F IRESite_ID:587, pGem-LamB1-F IRESite_Id:588).
Citations:
Petz M., Kozina D., Huber H., Siwiec T., Seipelt J., Sommergruber W., Mikulits W. (2007) The leader region of Laminin B1 mRNA confers cap-independent translation. Nucleic Acids Res. 35(8):2473-2482
IRESs:
IRES:
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  LamB1_-335_-1
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1211-1545
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  57
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  391
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -391
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -57
The sequence of IRES region aligned to its secondary structure (if available):


Citations:
Petz M., Kozina D., Huber H., Siwiec T., Seipelt J., Sommergruber W., Mikulits W. (2007) The leader region of Laminin B1 mRNA confers cap-independent translation. Nucleic Acids Res. 35(8):2473-2482
The translation experiments:
Translation results:
IRESite Translation Id: 621
Version: 2 Last change: 2009-04-30 11:36:54
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 MIM-R
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  142.000
Name of IRES used as the positive control:
  EMCV-R
Name of the plasmid used as the positive control.
pR-EMCV-F
Name of the plasmid used as the negative control.
pR-F
IRESite Id of the plasmid used as positive control.
  585
IRESite Id of the plasmid used as negative control.
  584
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  25.000
The size (length) of intercistronic region in the positive control:
769
The size (length) of intercistronic region in the negative control:
112
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:
Measured luciferase activity values were provided by M. Petz for Figure 3C.

Additional notes from M Petz:

Concerning Fig. 3A, I have checked the data again. The experiment was repeated several times and the ß-Gal
activities of the cotransfected plamid were always quite equal. The normalisation to the ß-gal values did
not affect the ratio of the firefly activities in any significant way.
Citations:
Petz M., Kozina D., Huber H., Siwiec T., Seipelt J., Sommergruber W., Mikulits W. (2007) The leader region of Laminin B1 mRNA confers cap-independent translation. Nucleic Acids Res. 35(8):2473-2482
Translation results:
IRESite Translation Id: 622
Version: 1 Last change: 2009-04-30 11:36:54
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 SW480
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  208.000
Name of IRES used as the positive control:
  EMCV-R
Name of the plasmid used as the positive control.
pR-EMCV-F
Name of the plasmid used as the negative control.
pR-F
IRESite Id of the plasmid used as positive control.
  585
IRESite Id of the plasmid used as negative control.
  584
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  18.000
The size (length) of intercistronic region in the positive control:
769
The size (length) of intercistronic region in the negative control:
112
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:
Measured luciferase activity values were provided by M. Petz for Figure 3D.

Additional notes from M Petz:

Concerning Fig. 3A, I have checked the data again. The experiment was repeated several times and the ß-Gal
activities of the cotransfected plamid were always quite equal. The normalisation to the ß-gal values did
not affect the ratio of the firefly activities in any significant way.
Citations:
Petz M., Kozina D., Huber H., Siwiec T., Seipelt J., Sommergruber W., Mikulits W. (2007) The leader region of Laminin B1 mRNA confers cap-independent translation. Nucleic Acids Res. 35(8):2473-2482
Last change to the database: 2015-04-16 16:45:23 GMT+1