The nucleic acid data:
IRESite Id: 103 Version: 17
Originaly submitted by: Martin Mokrejš Submission date: 2006-01-11 00:00:00
Reviewed by: Martin Mokrejš Last change: 2009-09-01 18:39:29
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  our_best_guess
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  BCL2
The genetic origin of this natural mRNA/+RNA:
  nuclear
The GenBankId GI:# number of exactly this mRNA/+RNA sequence:
179366
Synonyms of the gene name:
Synonym: Bcl-2
The mRNA/+RNA description: 
Human B-cell leukemia/lymphoma 2 (bcl-2) proto-oncogene mRNA encoding bcl-2-alpha protein, complete cds.
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 organism containing this mRNA with IRES segment in its genome:
Homo sapiens
A promoter reported in cDNA corresponding to IRES sequence:
  yes
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:
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:
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:
not_tested
Cryptic promoter presence was confirmed by expression from a promoter-less plasmid:
weak_promoter_confirmed
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 HEK 293T/17 (ATCC CRL-11268)
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:
BCL2
The description of the protein encoded in this ORF:
BCL2 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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1459-2178
Remarks:
The sequence of the BCL2 mRNA (1458b 5'-UTR, 5086b total) corresponds to the sequence in Fig. 3A in Tsujimoto
and Croce, 1986 which has in addition to the GenBank record also the polyA tail stretch. According to Sherrill
et al., 2004, this mRNA sequence corresponds to the so called 5.5 kb variant and should originate from the P1
promoter. The 1458b long 5'-UTR contains an intron, which is only optionally spliced out in vivo.
Excision of the intron does not affect IRES activity (K. Sherrill, personal communication).

BTW, the 3.5kb variant (146b 5'-UTR, 911b total) is stored under GI:179368 and is in Tsujimoto and Croce, 1986
in Fig. 3B.

Back to Sherrill et al., 2004, the transcript integrity was verified by RT-PCR. Cells were transfected by DNA
and also directly by in vitro transcribed mRNA. RNAi targeted against renilla luciferase in the first cistron
was contransfected into the cells to lower background noise by ~80%. Possible promoter presence within part of
the BCL2 5'-UTR studied was examined by use of a promoter-less plasmid (Fig. 3A) and also judged from the
effect of RNAi on levels of Rluc and Fluc (although such result could be biased by the fact aberrant splicing
was detected as pointed above it seems excision of the intron does not affect IRES activity; K. Sherrill,
personal communication). In case of BCL2 and XIAP IRESs it was found that RNAi decreased RLuc luciferase but
not FLuc activity in same scale (Fig. 3B). The Northern blot analysis and RNAi assay confirmed the BCL2 region
studied had a weak promoter activity (should be the P2 promoter described elsewhere?). The experiments have
shown/confirmed that the pRL-BCL2-FL plasmid contains previously known 3' splice acceptor site within the BCL2
region. The presence of the acceptor site resulted in both bicistronic Rluc-BCL2-Fluc mRNA and also Fluc
monocistronic mRNA being produced in vivo. Note: The IRES region studied by Sherrill et al., 2004 was only
1138b upstream the START codon -- instead of 1146b upstream as misreported in the article (K. Sherrill,
personal communication) because longer region could not be amplified by RT-PCR, possibly due to the very high
GC content.

Additional comments from K. Sherrill:

A key point is that our evidence for BCL-2 IRES activity is based exclusively
upon transfection of mRNA transcripts containing the BCL-2 IRES 5'UTR, and not
DNA transfection. Our RT-PCR results and RNAi work were performed only to
show the dangers inherent with defining an IRES solely by transfecting DNA
alone. The minor BCL-2 promoter that is present within this sequence is not
cryptic, but rather well-defined in the literature. However, it is primarily
active in lymphocytes. There is nothing to be taken "with a grain of salt"
as these data are not the supporting data of the paper. They simply show why
we had to obtain all of our REAL data via direct transfection of capped and
poly-adenylated mRNA, which is to avoid the possibility that promoter and/or
splicing activity might have affected our data.

In addition, I'd like to note that the presence (or absence) of the
alternatively-spliced intron within this BCL-2 5'UTR doesn't appear to
affect IRES activity, as we selectively deleted this intron and saw no
apparent effect.


Cammas et al. (2007) used the Bcl2 IRES in their RNAi experiments showing that either the splicing issue or
the promoter make the RNAi-based approach not unsuccessful (Supplemental Figure S2 of their publication).
Citations:
Tsujimoto Y., Croce C. M. (1986) Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc. Natl. Acad. Sci. U. S. A. 83(14):5214-5218
Sherrill K. W., Byrd M. P., Van Eden M. E., Lloyd R. E. (2004) BCL-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279(28):29066-29074
Cammas A., Pileur F., Bonnal S., Lewis S. M., Leveque N., Holcik M., Vagner S. (2007) Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs. Mol. Biol. Cell. 18(12):5048-5059
IRESs:
IRES:
Version: 4 Last change: 2007-01-23 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  BCL2
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  322-1458
Conclusion:
  weakly_supported_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
The BCL2 IRES has 6% activity of the HCV IRES in direct mRNA transfection (capped transcripts and 35bp long
polyA in human HEK 293T cells).
Citations:
Sherrill K. W., Byrd M. P., Van Eden M. E., Lloyd R. E. (2004) BCL-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279(28):29066-29074
Tsujimoto Y., Croce C. M. (1986) Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc. Natl. Acad. Sci. U. S. A. 83(14):5214-5218
Last change to the database: 2015-04-16 16:45:23 GMT+1