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 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 the putative Rbm3 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 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): 7775-8494
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 (-267bp upstream of the cellular initiator
AUG codon): tataatttcttcttccagAA.
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). More details
in Figure 3 and Supplementary Information 12 of the article by Baranick et al. (2008). They report that the
sequence reported originally as Rbm3 IRES comes actually from Thoc1 mRNA (AY052560) and not from Rbm3 (Suppl.
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 rescued as expected (mutant 2 in Supplementary Figure 10). However, the
mutations in mutants 1 and 3 in regions elsewhere should have not rescued the viral production but did quite
well in case of mutant 3. 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.