The nucleic acid data:
IRESite Id: 559 Version: 4
Originaly submitted by: Martin Mokrejš Submission date: 2009-03-13 17:27:33
Reviewed by: Martin Mokrejš Last change: 2009-09-02 22:13:19
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: 
Putative in vivo transcript driven from CMV IE promoter and terminated after the SV40 late poly(A) signal
containing GLuc (Gaussia luciferase) and GFP cistrons separated by the putative eIF4G IRES.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the plasmid:
pGLuc-eIF4G-GFP
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?):
  yes
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  yes
A promoter reported in cDNA corresponding to IRES sequence:
  not tested
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:
heterogeneous_population_of_molecules_found
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:
eIF4G
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:


GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pGLuc-eIF4G-GFP.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:
GLuc
The description of the protein encoded in this ORF:
Gaussia 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):
  91-648
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
GFP
The description of the protein encoded in this ORF:
green fluorescent protein
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):
  1028-1747
Remarks:
In addition to the unspliced message 4 alternatively spliced products were reported in HeLa cells transiently
transfected by this plasmid. Although the authors intended to use a plasmid which would be depleted of
5'-splice donor sites (Supplementary Figure 6) still several 5'-splice donor sites remained in GLuc coding
region and even in the eIF4G sequence itself. Please refer to the Supplementary Figure 7 in Baranick et al.
(2008).


The sequences around the 5'-splice donor were:

TGCTGGTGGGgtagggatga (in the eIF4G sequence itself)

GGGCCCCAGGgtgtgcagcc (in the eIF4G sequence itself, intron emphasized in lowercase letters of this IRESite
record)

CCAGGGCCAAgtcgataaga (in GLuc coding region, C-terminus)

ACCTTCGCAAgcaagatcca (in GLuc coding region, C-terminus, also functional in XIAP-based plasmid IRESite_Id:563)


The 3'-splice acceptor region within eIF4G was: ttatagctttctttccccagAT




Comments from IRESite curator:

The first splice variant has intercistronic region of size 122bp (instead of 379bp as the unspliced message)
while both reported ORFs intact.

The second splice variant has intercistronic region of size 88bp (instead of 379bp as the unspliced message)
while both reported ORFs intact.

The third splice variant has the first cistron merged in another frame to the second cistron, quite
surprisingly the fusion protein extends even behind the GFP protein ORF by 17 bp. Scanning ribosome would have
to bypass AUGs of 6-10 ORFs 538-720bp to synthesize GFP protein (of these are 2 uORFs).

The fourth splice variant has modified C-terminal region of the first cistron spanning the eIF4GI 5'-UTR
insert sequence and hits a STOP codon in the very beginning of GFP ORF. However, in another ORF there could be
produced GFP protein in its original reading frame with extra 26 aminoacid residues fused to it N-terminus.
And, obviously unaltered GFP protein could be produced as well. Scanning ribosome would have to bypass AUGs of
6-9 ORFs over a distance of 538-644bp to synthesize GFP protein (of these are 3 uORFs).
Citations:
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
IRESs:
IRES:
Version: 2 Last change: 2009-09-02 22:13:19
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  eIF4G_-368_-12
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  665-1021
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  17
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  373
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -363
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -7
The sequence of IRES region aligned to its secondary structure (if available):


Citations:
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
The translation experiments:
Translation results:
IRESite Translation Id: 601
Version: 1 Last change: 2009-07-16 14:37:08
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:
  21.700
Name of IRES used as the positive control:
  EMCV-R_-541_-7
Name of the plasmid used as the positive control.
pGLuc-EMCV-GFP
Name of the plasmid used as the negative control.
pGLuc-GFP
IRESite Id of the plasmid used as positive control.
  557
IRESite Id of the plasmid used as negative control.
  556
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  10.800
The size (length) of intercistronic region in the positive control:
565
The size (length) of intercistronic region in the negative control:
17
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 1B.
Citations:
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
Translation results:
IRESite Translation Id: 602
Version: 1 Last change: 2009-07-16 14:37:08
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 NIH/3T3 (ATCC CRL-1658)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  16.600
Name of IRES used as the positive control:
  EMCV-R_-541_-7
Name of the plasmid used as the positive control.
pGLuc-EMCV-GFP
Name of the plasmid used as the negative control.
pGLuc-GFP
IRESite Id of the plasmid used as positive control.
  557
IRESite Id of the plasmid used as negative control.
  556
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  10.900
The size (length) of intercistronic region in the positive control:
565
The size (length) of intercistronic region in the negative control:
17
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 1B.
Citations:
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
Last change to the database: 2015-04-16 16:45:23 GMT+1