The nucleic acid data:
IRESite Id: 110 Version: 14
Originaly submitted by: Martin Mokrejš Submission date: 2006-01-12 00:00:00
Reviewed by: Martin Mokrejš Last change: 2015-04-08 23:31:45
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  hopefully_full-length_mRNA
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  Apaf-1
The genetic origin of this natural mRNA/+RNA:
  nuclear
The GenBankId GI:# number of the most similar mRNA/+RNA sequence to this one.
164692476 
The mRNA/+RNA description: 
Homo sapiens apoptotic protease activating factor 1 (Apaf-1) mRNA, complete cds. Similar to C. elegans cell
death gene ced-4.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  only_fragment_published_or_from_author_and_the_rest_is_a_guess
The organism containing this mRNA with IRES segment in its genome:
Homo sapiens
A promoter reported in cDNA corresponding to IRES sequence:
  unclear
The total number of notable open-reading frames (ORFs):
  1
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:
not_tested
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
not_tested
Integrity (uniformity) of mRNA tested using RNAi:
heterogeneous_population_of_molecules_found
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)
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 3 Last change: 2009-09-01 19:57:05
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Apaf-1
The description of the protein encoded in this ORF:
apoptotic protease activating factor 1
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):
  737-4354
Remarks:
The best really existing mRNA sequence in Genbank is AK307509 (GI:164692476) which has longer 5'-UTR by 159bp
(obtained by oligo-capping approach) compared to entry AF013263 (GI:2330014). The below mentioned 7kb long
sequence is a chimera of Apaf1 mRNA with another gene since position 5244:


>emb|CR603841.1| UniGene info full-length cDNA clone CS0DI077YE15 of Placenta Cot 25-normalized of Homo
sapiens Length=1596

AF013263  5244  GGTCTTGAACTCTTGGCCTCAAGTAATCCTCCTGCCTCAGCCTCCCAAAGTGTTGGGATT  5303
                ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct     1     GGTCTTGAACTCTTGGCCTCAAGTAATCCTCCTGCCTCAGCCTCCCAAAGTGTTGGGATT  60

[cut]

AF013263  6384  TCCTTAGAATACCCAAATCATAATTTTATTTGTACACACTGTTAGGGGCTCATCTCATGT  6443
                |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct     1141  TCCTTAGAATACCCAAATCATAATTTTATTTGTACACACTGTTAGGGGCTCATCTCATG-  1199

AF013263  6444  AGG  6446
                |||
Sbjct     1200  AGG  1202

[cut]

AF013263  6617  GAGGATTCCTTGAGCCCTGGAGTTTGAGTCCAGCCTGGGTGACATAGCAAGACCCTGTCT  6676
                ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct     1199  GAGGATTCCTTGAGCCCTGGAGTTTGAGTCCAGCCTGGGTGACATAGCAAGACCCTGTCT  1258

[cut]

AF013263  6977  gaatttaaaaaatttttgtaaaaataaaattcacaaaa  7014
                ||||||||||||||||||||||||||||||||||||||
Sbjct     1559  GAATTTAAAAAATTTTTGTAAAAATAAAATTCACAAAA  1596




The Apaf1-mRNA region in this chimeric message of AF013263 ends at the position 4283:

>gb|EF560718.1| UniGene infoGene info Homo sapiens clone DKFZp781B1145 APAF1 protein (APAF1) mRNA, complete
cds Length=3744

AF013263   573  GGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGG  632
                ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct      1    GGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGG  60

[cut]

AF013263  4231  TGATATCAACTTTTTATAAAGCTCTTAATTGTTGTGCAGTATTGCATTCATTA  4283
                |||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct     3692  TGATATCAACTTTTTATAAAGCTCTTAATTGTTGTGCAGTATTGCATTCATTA  3744




Interestingly, several mRNAS incomplete at the 3'-end terminate just at position 4162 which is a STOP codon:

>emb|AJ243004.1| UniGene infoGene info Homo sapiens mRNA for apoptotic protease activating factor 1
Length=3618

[cut]

AF013263  4111  TGTGACTGTGGATAATCTTGGTATTTTATATATTTTACAGACTTTAGAATAA  4162
                ||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct     3567  TGTGACTGTGGATAATCTTGGTATTTTATATATTTTACAGACTTTAGAATAA  3618



In conclusion, we have used sequence in AK307509 for the 5'-UTR and most of the CDS and the record EF560718
to get up to the 3'-end.


Several alternatively spliced messages exist but the splice junctions are in the CDS, thus not important to
the IRES story.




Integrity of the transcripts was verified by Northern blot using randomly primed cDNA probes against Fluc.
Shorter transcript fragment of 1.3kb was detected (also found in pRmycF transcripts containing c-myc). No more
sensitive testing of integrity of transcripts produced was done, e.g. RT-PCR of the in vivo produced
transcription products or direct mRNA transfection or tests based on a promoter-less plasmid. [Coldwell et
al., 2000]


It appears the very two 3'-most bases of the 5'-UTR of Apaf-1 were not cloned into the pRAF plasmid vector
because they had to be replaced by 'cc' bases of the cloning NcoI site 'ccATGg'. Please refer to the
annotation of pRAF plasmid (IRESiteID:341).

Cammas et al. (2007) confirmed either a splicing or cryptic promoter issue with the pRAF plasmid in DNA
transfections (Supplemental Figure S2) in RNAi based assay in which they were not able to inhibit the Apaf-1
IRES activity like of EMCV, HRV IRES. They repeated results of Sherril et al. (2004) showing that either a
splicing issue or a promoter is functional in HeLa cells when Bcl2 IRES is placed into the pRF vector.
Further, they show that in direct mRNA transfection Apaf-1 IRES activity is only 2x above the empty vector
control (capped, poly(A)-tailed messages) and thus conclude as not functional.
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
Cammas A., Pileur F., Bonnal S., Lewis S. M., Leveque N., Holcik M., Vagner S. (2007) Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs. Mol. Biol. Cell. 18(12):5048-5059
IRESs:
IRES:
Version: 7 Last change: 2009-09-01 19:57:05
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  Apaf-1
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  504-734
Conclusion:
  putative_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):



Remarks:
The -233 to -1 (due to the cloning approach possibly only -233 to -3) fragment retains 75% activity (Fig. 6B)
of the full-length 5'-UTR (1-577b). Apaf-1 IRES is functional in HeLa, HepG2 cells, lower activity was seen in
MCF7, HK293, COS7, MRC5 cells. No activity seen in SY5Y and Balb/c cells.
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
RNA:protein interactions:
The RNA:protein interaction:
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The description of the protein interacting with the RNA:
Heterogeneous nuclear ribonucleoprotein (hnRNP) A1
The organism where this RNA:protein interaction occurs:
Homo sapiens HeLa (ATCC CCL-2)
Remarks:
The interaction was confirmed by several methods using HeLa cell nuclear extracts (NE) with 32P-labeled RNAs.
Data in Figure 1.
Citations:
Cammas A., Pileur F., Bonnal S., Lewis S. M., Leveque N., Holcik M., Vagner S. (2007) Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs. Mol. Biol. Cell. 18(12):5048-5059
Regions with experimentally determined secondary structures:
A region with the experimentally determined secondary structure:
IRESite 2D Struct Id: 26
Version: 1 Last change: 2009-09-01 19:57:05
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The function of the 2D structure:
IRES
The 2D structure causes frameshift:
no
The absolute position of the experimentally mapped region (the range includes START and STOP codons or their equivalents):
160-739
The underlying nucleic acid sequence and structure of the mapped region:



Remarks:
Structure from Fig. 4E.
4.1.1. Enzymes used to characterize at least partially the 2D structure.
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
ss_experiment_with_enzyme_id: 48
The temperature (in degrees of Celsia):
22
The enzymatic method used to determine the 2D structure:
ribonuclease V1
Enzyme or a combination of enzymes used in a single experiment with respective buffer:
Version: 0
pH
7.00
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
100.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
120.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
4.1.2. Chemicals used to characterize at least partially the 2D structure.
Chemical reagent used with its respective buffer:
ss_experiment_with_chemical_id: 20
The temperature (in degrees of Celsia):
0
The chemical reagent used to determine the 2D structure:
DMS
Chemical reagent used with its respective buffer:
Version: 0
pH
7.00
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
100.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
100.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Other buffer components and their relative concentrations:
20mM acetate (CH3COO)-
Chemical reagent used with its respective buffer:
ss_experiment_with_chemical_id: 21
The temperature (in degrees of Celsia):
0
The chemical reagent used to determine the 2D structure:
kethoxal
Chemical reagent used with its respective buffer:
Version: 0
pH
7.00
Li+ [mM]
0
Na+ [mM]
0
K+ [mM]
100.00
Mg2+ [mM]
10.00
Ca2+ [mM]
0
Cl- [mM]
100.00
Tris [mM]
10.00
BSA [mM]
0
HEPES [mM]
0
EGTA [mM]
0
EDTA [mM]
0
cacodylate [mM]
0
Other buffer components and their relative concentrations:
20mM acetate (CH3COO)-
Citations:
Mitchell S. A., Spriggs K. A., Coldwell M. J., Jackson R. J., Willis A. E. (2003) The Apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with PTB and unr. Mol. Cell. 11(3):757-771
Last change to the database: 2015-04-16 16:45:23 GMT+1