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
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
AF013263 5244 GGTCTTGAACTCTTGGCCTCAAGTAATCCTCCTGCCTCAGCCTCCCAAAGTGTTGGGATT 5303
Sbjct 1 GGTCTTGAACTCTTGGCCTCAAGTAATCCTCCTGCCTCAGCCTCCCAAAGTGTTGGGATT 60
AF013263 6384 TCCTTAGAATACCCAAATCATAATTTTATTTGTACACACTGTTAGGGGCTCATCTCATGT 6443
Sbjct 1141 TCCTTAGAATACCCAAATCATAATTTTATTTGTACACACTGTTAGGGGCTCATCTCATG- 1199
AF013263 6444 AGG 6446
Sbjct 1200 AGG 1202
AF013263 6617 GAGGATTCCTTGAGCCCTGGAGTTTGAGTCCAGCCTGGGTGACATAGCAAGACCCTGTCT 6676
Sbjct 1199 GAGGATTCCTTGAGCCCTGGAGTTTGAGTCCAGCCTGGGTGACATAGCAAGACCCTGTCT 1258
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
AF013263 573 GGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGG 632
Sbjct 1 GGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAGCTCTGGAAAAGG 60
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
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
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.
The IRES absolute position (the range includes START and STOP codons or their equivalents): 504-734
How IRES boundaries were determined: experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):
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.