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: 3UTR_possibly_incomplete
The mRNA/+RNA description:
Putative in vivo transcript of plasmid with chimeric MoMLV provirus driven from CMV IE promoter. The capped
and unspliced viral transcript encodes gag/gag-pol and GFP proteins separated by XIAP IRES.
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 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: heterogeneous_population_of_molecules_found
The description of the protein encoded in this ORF: green fluorescence 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): 8024-8743
Remarks:
Presence of alternative splice products was monitored functionally by evaluation of efficiency of the virus
to replicate, produce reverse-transcriptase which activity in the media was measured, and infectious virions.
In this case alternatively spliced monocistronic products were found by RT-PCR containing only the GFP ORF
intact. It is presumed that these messages contributed the most to the observed GFP activity.
The splice acceptor site located in the putative IRES region was (-33bp upstream of the cellular initiator
AUG codon): ataatgttctctttttagAA.
The 5'-splice donors were located:
- in the non-coding region after U5 and well before gag CDS (788-GT-789 of this proviral plasmid, actually
206-gt-207 of the putative non-spliced mRNA)
- in env gene GTATGTCGGgtatggctg (6634-GT-6635 of this proviral plasmid, actually 6052-gt-6053 of the
putative, non-spliced mRNA)
The plasmid flatfile attached to this entry has the largest intron in lowercase letters although shorter
splice variants existed as well (also the cryptic 5'-splice donor within env gene was used). Moreover, the
3'-splice acceptor site appears to be used in cellular XIAP transcripts as well. More details in Figure 3
and Supplementary Information 12 of the article by Baranick et al. (2008).
From: C. Logg
Date: Mar 5 2009
The provirus sequence is almost all derived from a clone of Moloney MLV (NC_001501). We switched the ecotropic
env gene with the amphotropic env gene from MLV 4070A (M33469), so it's a hybrid of those two MLV strains. In
MoMLV, there is a stop codon at the end of gag, which is sometimes bypassed. In these cases, a gag-pol
polyprotein is produced, and this is how pol is translated. Pol doesn't have a separate start codon. I believe
all of the sequences are correct. We've sequenced the entire virus-derived portions of the original provirus
plasmids from which all of the other provirus plasmids were constructed. For each of these individual later
plasmids, we've at least verified the IRES/UTR/control-GFP insert sequence. I am assuming that no mutations
outside that region were acquired during the cloning. Each of the provirus plasmids has either a BsiWI or MluI
at the 5' side of the IRES (just after the env stop codon) and a NotI at the 3' side of GFP (just before the
MLV 3' UTR). These are the sites we used to transfer the IRES-GFP cassettes.
When the acceptor site was destroyed by mutation the activity of the reverse transcriptase produced into the
cultivation media by the virus was NOT rescued contrary to expectations (mutant 1 in Supplementary Figure 10).
Further in contrast, the mutations in mutants 2, 3, 4 in regions elsewhere should have not rescued the viral
production but did quite well in case of mutants 3 and 4. Thus, the results are a bit puzzling. Maybe due to
additional mutations outside the region sequenced?
However, the GFP results based on these mutants shown in Figure 4 reflect as stated by the authors that the
type I splicing case (shown in black in Figure 4A) is proportional to the GFP values shown in Figure 4B.
This plasmid has env - MluI - eIF4GI - BsiWI - GFP - NotI. In contrast to the other vectors has an extra BsiWI
site (like the vector with eIF4GI insert). It is not clear whether the sequence provided by C. Logg is wrong
or the description just does not match this particular case.
The IRES absolute position (the range includes START and STOP codons or their equivalents): 7748-8017
How IRES boundaries were determined: experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF: 1911
3'-end of IRES relative to last base of the STOP codon of the upstream ORF: 2180
5'-end of IRES relative to first base of the START codon of the downstream ORF: -276
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):
Remarks:
The sequence inserted as the XIAP IRES is derived from EST sequence and seems not being covered by NCBI
NonRed nor RefSeq. However, aligns to the 3'-most region the 5'-UTR studied by M. Holcik so should be
authentic:
[the alignment was arbitrarily cut here]
holcik TATAATATATTCTGCATCACAGTTTACATATTTATGTAAAATAAGCATTTAAAAATTATT
baranick ---------------------------------------------------------ATT
***
holcik AGTTTTATTCTGCCTGCTTAAATATTACTTTCCTCAAAAAGAGAAAACAAAAATGCTAGA
baranick AGTTTTATTCTGCCTGCTTAAATATTACTTTCCTCAAAAAGAGAAAACAAAAATGCTAGA
************************************************************
holcik TTTTACTTTATGACTTGAATGATGTGGTAATGTCGAACTCTAGTATTTAGAATTAGAATG
baranick TTTTACTTTATGACTTGAATGATGTGGTAATGTCGAACTCTAGTATTTAGAATTAGAATG
************************************************************
holcik TTTCTTAGCGGTCGTGTAGTTATTTTTATGTCATAAGTGGATAATTTGTTAGCTCCTATA
baranick TTTCTTAGCGGTCGTGTAGTTATTTTTATGTCATAAGTGGATAATTTGTTAGCTCCTATA
************************************************************
holcik ACAAAAGTCTGTTGCTTGTGTTTCACATTTTGGATTTCCTAATATAATGTTCTCTTTTTA
baranick ACAAAAGTCTGTTGCTTGTGTTTCACATTTTGGATTTCCTAATATAATGTTCTCTTTTTA
************************************************************
holcik GAAAAGGTGGACAAGTCCTATTTTCAAGAGAAG
baranick GAAAAGGTGGACAAGTCCTATTTTCAA------
***************************
Baranick et al. postulate that the "IRES" activity observed so far was due to the splice artifacts.