The nucleic acid data:
IRESite Id: 66 Version: 18
Originaly submitted by: Petra Sekyrová Submission date: 2005-08-10 00:00:00
Reviewed by: Martin Mokrejš Last change: 2007-07-24 00:00:00
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:
  Hairless
The genetic origin of this natural mRNA/+RNA:
  nuclear
The GenBankId GI:# number of exactly this mRNA/+RNA sequence:
578331
The mRNA/+RNA description: 
D. melanogaster mRNA for hairless serine rich protein. The third ORF product of 120kDa is produced from 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 organism containing this mRNA with IRES segment in its genome:
Drosophila melanogaster
A promoter reported in cDNA corresponding to IRES sequence:
  no
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:
homogeneous_population_of_molecules_confirmed
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:
no_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:
not_tested
The organism used:
Drosophila melanogaster S2
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 1 Last change: 2006-08-01 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
hairless-120
The description of the protein encoded in this ORF:
hairless serine rich protein initiated from M3 AUG(704) having 120kDa
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:
  yes
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  704-3496
OPTIONAL: The function of the encoded protein
Notch antagonist, silences Notch target genes by binding to Su(H) and thereby transforming Su(H) from an
activator to a supressor. D. melanogaster gene Hairless, abbreviated as H, is reported here. It encodes a
product with transcription corepressor activity involved in sensory organ determination which is a component
of the nucleus; it is expressed in the embryo (anterior embryonic/larval midgut, central nervous system,
cephalic furrow, embryonic central nervous system and 5 other listed tissues), larva (imaginal disc), ovary
(nurse cell) and prepupa and pupa (epithelial cell and tormogen cell). It has been sequenced and its amino
acid sequence is also available. It has been mapped by recombination to 3-69.5 and cytologically to 92F3. It
interacts genetically with Su(H), Dl, neur, Egfr, gro and 66 other listed genes. (www.flybase.org)
Remarks:
Two H protein isoforms are produced during development, whereby the shorter is generated by internal
translation initiation preferentially during mitosis. The two isoforms are both indispensable for normal fly
development, suggesting a requirement for H protein throughout the cell cycle to antagonize Notch signaling
properly. Hypotheses: H might act as a homo- or heterodimer (or multimer) involving the two isoforms, and
heteromers might be functionally distinct from homomers. H activity might also be required during times when
normal translation is inhibited, like in cellular stress situations and/or during the mitotic phase of the
cell cycle. The Hairless gene contains 3 start codons: M1 (263. nt) does not conform well with the translation
initiation consensus site for Drosophila genes in contrast to the M2 codon (M1 is probably less often used for
as translation start site, resulting in 150 kDa protein). M2 (317. nt) serves as a start codon for production
of longer version more often (is in more favorable context), still 150 kDa protein is yielded. Third start
codon is located at 704. nt and start of translation from there results in shorter isoform (120 kDa). It is
postulated internal initiation of translation occurs between M2 and M3.



The IRES was tested in transiently transfected cells using bicistronic constructs. Promoter-less plasmid BTI
(Bluescript based, with M2 start codon mutated to Leu) has been used to proof at least no strong promoter is
present. T3 transcripts give rise in rabbit reticulocyte lysates to both Hairless protein forms. Promoter-less
plasmid test based on pBluescript plasmid has shown no convincing promoter activity (Fig. 2B), or at least no
strong promoter is present. Leaky scanning in this case can again be excluded by the fact that mutation of
boxA (UUUUU) prevents Hp120 production during in vitro translation (Fig. 3C, detection by antibodies).
Finally, ectopic induction of the WT transcripts from a heat-inducible promoter gives rise to both isoforms in
the expected ratio (Fig. 1B; ref. 6). In summary, the putative IRES contains UUUUU stretch which is necessary
for its function.
Citations:
Maier D., Nagel A. C., Preiss A. (2002) Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation. Proc. Natl. Acad. Sci. U. S. A. 99(24):15480-15485
IRESs:
IRES:
Version: 10 Last change: 2007-01-11 00:00:00
Originaly submitted by: Petra Sekyrová Reviewed by: Martin Mokrejš
The IRES name:
  hairless
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  308-742
Conclusion:
  putative_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
The hairless IRES sequence contains a penta-uridine box approx. 30 nt upstream of the M3 start codon embeded
in a GC-rich sequence. This structure is reminiscent of the picorna virus and ODC IRESs and indeeed seems to
be essential for hairless IRES activity: changing uridines into adenines inhibited translation indicating that
this box is essential sequence element for the activity of hairless IRES.

The function of IRES element was confirmed in cultured Drosophila S2 cells and also in organs dissected from
transgenic flies.
Citations:
Maier D., Nagel A. C., Preiss A. (2002) Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation. Proc. Natl. Acad. Sci. U. S. A. 99(24):15480-15485
Last change to the database: 2015-04-16 16:45:23 GMT+1