The nucleic acid data:
IRESite Id: 569 Version: 5
Originaly submitted by: Martin Mokrejš Submission date: 2009-03-16 15:57:28
Reviewed by: Martin Mokrejš Last change: 2009-04-08 19:29:22
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 name of the plasmid:
MoMLV-provirus-Rbm3-GFP
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 organism used:
Homo sapiens PC-3 (ATCC CRL 1435)
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
Rbm3
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Mus musculus
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
MoMLV-provirus-Rbm3-GFP.jpg
The total number of notable open-reading frames (ORFs):
  3
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
gag
The description of the protein encoded in this ORF:
gag polyprotein
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):
  621-2237
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
gag-pol
The description of the protein encoded in this ORF:
gag-pol polyprotein
The translational frameshift (ribosome slippage) involved:
  0
The ribosome read-through involved:
  yes
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):
  621-5837
ORF
ORF position:   3
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
GFP
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
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 (-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.
Image 11).




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.
Citations:
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
IRESs:
IRES:
Version: 2 Last change: 2009-03-16 16:37:38
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  Rbm3_22nt_module
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  7747-7768
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  1910
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  1931
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -28
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:
Baranick et al. postulate that the "IRES" activity observed so far was due to the splice artifacts.
Citations:
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
The translation experiments:
Translation results:
IRESite Translation Id: 609
Version: 1 Last change: 2009-07-16 15:13:35
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HeLa (ATCC CCL-2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  302.000
Name of IRES used as the positive control:
  EMCV-R_-541_-7
Name of the plasmid used as the positive control.
MoMLV-provirus-EMCV-GFP
Name of the plasmid used as the negative control.
MoMLV-provirus-GFP
IRESite Id of the plasmid used as positive control.
  550
IRESite Id of the plasmid used as negative control.
  549
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  9.000
The size (length) of intercistronic region in the positive control:
2458
The size (length) of intercistronic region in the negative control:
1914
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
Data from Figure 2B.
Citations:
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
Translation results:
IRESite Translation Id: 610
Version: 1 Last change: 2009-07-16 15:13:35
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens PC-3 (ATCC CRL 1435)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  394.000
Name of IRES used as the positive control:
  EMCV-R_-541_-7
Name of the plasmid used as the positive control.
MoMLV-provirus-EMCV-GFP
Name of the plasmid used as the negative control.
MoMLV-provirus-GFP
IRESite Id of the plasmid used as positive control.
  550
IRESite Id of the plasmid used as negative control.
  549
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  9.000
The size (length) of intercistronic region in the positive control:
2458
The size (length) of intercistronic region in the negative control:
1914
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
Data from Figure 2B.
Citations:
Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738
Last change to the database: 2015-04-16 16:45:23 GMT+1