The cloned 5'-UTR by van Eden et al. (2004) region was not a complete 5'-UTR. There are 3 mutations in
the cloned sequence provided by R. Lloyd in comparison to both German and NIH cDNA projects: GI:34367137 and
GI:22382083. -297nt from initiator ATG is A instead of G, -177nt is A instead of T, -76nt is G instead of T.
The sequence provided to IRESite by R. Lloyd spans -1115 to +4 (while the +4 base not shown in van Eden
et al. (2004) Fig. 1A is 'G'. van Eden showed already published 5'-UTR of NM_001166.2 (GI:10880127) GenBank
record which is 1159b long whereas current,, updated record GI:41349435 (NM_001166.3) contains 1.4kb
untranslated region. Thus, the sequence presented here in IRESite is longer by 240 nt at the very 5'-end
compared to the sequence shown by van Eden in Figure 1.
It appears the ATG codons at positions 1400 and 1463 are in-frame. The TAA stop codon at postion 1453 of the
previously reported uORF (Lewis et al., (2005), Fig. 2) is not in-frame with the ATG codon and thus it appears
based on the current sequence that the uORF does not exist. As a consequence, the IRES reported by M. Holcik
group spans into the CDS region whereas the region studied by R. Lloyd group was within the 5'-UTR. BLASTN
search for ESTs returns matches which show the current sequence with no uORF is correct.
Aberrantly spliced mRNAs were detected by RT-PCR (van Eden et al. (2004) Fig. 4D) which were not detectable
by Northern blot. The splice junction was AAGAGAAAG/CTATCAAAC and the splice event removed part of the RLuc
and most of cIAP1 5'-UTR from the intercistronic region (only 26 bp remained immediately upstream of the
initiatior ATG codon). Similarly, monocistronic mRNAs containing the chimeric intron and the cIAP1 5'-UTR
spliced at the same position.
Possible presence of a cryptic promoter was studied in a promoter-less plasmid and only very weak FLuc
activity was detected (Fig. 4C). In contrast, the control pGL3-Basic vector also lacking an SV40 promoter
expressed much lower amount of FLuc activity.
IRES activity was confirmed by direct bicistronic mRNA transfection (Fig. 5) which was capped and
The IRES absolute position (the range includes START and STOP codons or their equivalents): 285-1399
How IRES boundaries were determined: experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):
IRES activity measurable only in cells directly transfected by RNA was at the best comparable to the activity
of HCV IRES and about 2x the activity of negative control (capped and poly(A)-tailed RNAs). More importantly,
the Fluc/RLuc ratio values were around 0.08 so the IRES-driven expression was very low compared to the