The nucleic acid data:
IRESite Id: 577 Version: 10
Originaly submitted by: Martin Mokrejš Submission date: 2009-04-09 01:43:28
Reviewed by: Martin Mokrejš Last change: 2009-09-05 13:11:04
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  3UTR_incomplete
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  FeLV-Notch2
The genetic origin of this natural mRNA/+RNA:
  viral
The GenBankId GI:# number of the most similar mRNA/+RNA sequence to this one.
1622786 
The mRNA/+RNA description: 
Spliced subgenomic FeLV-Notch2 transcript with env gene replaced with part of Notch2 gene transcribed from the
FeLV retroviral promoter. The transcript does not contain complete CDS region of the Notch2 protein but only
the intracellular domain (ICD).
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  only_fragment_published_or_from_author
The organism containing this mRNA with IRES segment in its genome:
Felis silvestris
A promoter reported in cDNA corresponding to IRES sequence:
  unclear
The total number of notable open-reading frames (ORFs):
  1
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:
heterogeneous_population_of_molecules_found
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:
not_tested
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:
homogeneous_population_of_molecules_confirmed
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:
not_tested
The organism used:
Homo sapiens HeLa (ATCC CCL-2)
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 4 Last change: 2009-04-10 21:13:14
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FeLV-Notch2
The description of the protein encoded in this ORF:
Intracellular domain of Notch2 protein fused with C-terminus from part of FeLV env gene.
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):
  817-2313
Remarks:
Based on EST data the transcription start site of FeLV is quite exact (blastn query finds sequences starting
at same or almost same location with well aligning region 28-457 of this spliced mRNA sequence) so this mRNA
is complete at the 5'-end. The sequence of the cloned region for putative IRES activity was reverse-engineered
by Adam Lauring from the lab of J. Overbaugh because the original sequence files could not be used due to
technical issues. The genomes of closest strains of FeLV from the same lab are: M19392, M87886, M18246,
M89997. Based on the alignment of the FeLV-Notch2 partial sequence and of the genome of FeLV on can infer
regions contained in the subgenomic env-Notch2 message: M18247:341-545,5670-6046 (this is same Rohn et al.
(1996), Figure 5, FeLV/N2-B). Then positions 5370-7010 of Notch2 are included and then again retroviral
M18247:7716-. It is not clear where is the transcription termination site in the feline FeLV 3'-LTR region
and therefore this IRESite record includes complete 3'-LTR instead of just its leftmost part.


Part of the Notch2 CDS was appended to sequence of the cloned fragment based on a consensus sequence
derived easily from GenBank records (submitted from the lab of J. Overbaugh during the past): U47642, U47643,
U47644, U47645 differing in a few nucleotides each. The record U47644 spans most into the 3'-end and is
actually referred to in Lauring et al. (2000) in "Plasmid construction and Transfections".

The CDS region of Notch2 within this mRNA contains (although more than 1600 aa residues are missing on its
left and the total translation product in human is 2472 aa residues!). The FeLV-Notch2 fusion is not complete
either on its C-terminus but instead fused to the sequence of env CDS again. The next sequence shows where
is the first initiator methionine:

SKVFLEIDNRQCVQDSDQCFKNTDAAAALLASHAIQGTLSYPLVSVVSESLTRKGTQLLYLLAVAVVIILFIILLGVIMAKRKRKHGSLWLPEGFTLRRDSSNHKRREP
VGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQPKKAKAENEALLSEEDDAIDRRPWTQQHLEAADIRRTPSLALTPPQAEQEVNVLDVNVRGPDGCTPLMLASLR
GGSSDISDEDKDREDSSANIITDLVYQGASLQAQTDRTGEMALHLAARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARMNDGTTP
LILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLKNGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH
DIVRLLDEYNVTPSPPGTVLTSALSPVICGPNRSFLSLKHTPMGKKPRRPNTKSAMPTSLPNLAKEAKDAKGSRRKKSLSDKGQLSESSVTLSPVDSLESPHTYVSDTTS

but here the putative translation product can start at the nearest in-frame AUG codon, thus the ORF is
817-2313 with translation product (the sequence in lowercase originates from C-terminus of FeLV env gene):
                                                                              MAKRKRKHGSLWLPEGFTLRRDSSNHKRREP
VGQDAVGLKNLSVQVSEANLIGSGTSEHWVDDEGPQPKKAKAENEALLSEEDDAIDRRPWTQQHLEAADIRRTPSLALTPPQAEQEVNVLDVNVRGPDGCTPLMLASLR
GGSSDISDEDKDREDSSANIITDLVYQGASLQAQTDRTGEMALHLAARYSRADAAKRLLDAGADANAQDNMGRCPLHAAVAADAQGVFQILIRNRVTDLDARMNDGTTP
LILAARLAVEGMVAELINCQADVNAVDDHGKSALHWAAAVNNVEATLLLLKNGANRDMQDNKEETPLFLAAREGSYEAAKILLDHFANRDITDHMDRLPRDVARDRMHH
DIVRLLDEYNVTPSPPGTVLTSALSPVICGPNRSFLSLKHTPMGKKPRRPNTKSAMPTSLPNLAKEAKDAKGSRRKKSLSDKGQLSESSVTLSPVDSLESPHTYVSDTTS
pvplvynpnflhygpltnptpnsplrpmhp*

The record U47644 contains Notch2 fragment. The downstream bases were shown in Rohn et al, 1996 in Figure 5
and thus were inferred from M18247.

However, according to the original article the Notch2-ICD protein is being expressed in vivo and thought to be
expressed from this spliced envelope-like mRNA via IRES (the Notch2 sequence replaced most of the env gene
and therefore only a short, 72bp long uORF exists in the 814 bp long 5'-UTR region as a remnant of the
original env gene).

The integrity of the transcripts was assessed by RT-PCR (Rohn et al., 1996), S1 nuclease protection (Lauring
et al., 2000, Figure 1B), Northern blot (Lauring et al., 2000, Supplemental Figure 1).
Citations:
Lauring A. S., Overbaugh J. (2000) Evidence that an IRES within the Notch2 coding region can direct expression of a nuclear form of the protein. Mol. Cell. 6(4):939-945
Rohn J. L., Lauring A. S., Linenberger M. L., Overbaugh J. (1996) Transduction of Notch2 in feline leukemia virus-induced thymic lymphoma. J. Virol. 70(11):8071-8080
IRESs:
IRES:
Version: 2 Last change: 2009-04-10 20:07:07
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  FeLV-Notch2
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  582-819
Conclusion:
  putative_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
The range corresponds to the range 580-814 mentioned in the article (Figure 3B). This region is actually the
CDS region of the Notch2 gene (the FeLV encoded region is 1-580, the sequence since 581 came from the Notch2
ORF).
Citations:
Lauring A. S., Overbaugh J. (2000) Evidence that an IRES within the Notch2 coding region can direct expression of a nuclear form of the protein. Mol. Cell. 6(4):939-945
Last change to the database: 2015-04-16 16:45:23 GMT+1