The nucleic acid data:
IRESite Id: 322 Version: 4
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2008-10-18 20:48:23
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  hopefully_full-length_mRNA
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  LINE-1
The genetic origin of this natural mRNA/+RNA:
  viral
The GenBankId GI:# number of the most similar mRNA/+RNA sequence to this one.
2731765 
The mRNA/+RNA description: 
Mouse L1 inserted into the Mus musculus glycine receptor beta-subunit gene. The L1 spastic mRNA corresponds to
region 1356-8876 of GI:2731765.
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:
Mus musculus
A promoter reported in cDNA corresponding to IRES sequence:
  no
The total number of notable open-reading frames (ORFs):
  2
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:
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:
Mus musculus
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:
ORF1
The description of the protein encoded in this ORF:
Mouse L1 ORF1p, 43kDa, homotrimer, nucleic acid chaperone with RNA and ssDNA binding capability, essential for retrotransposition.
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):
  1787-2902
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
ORF2
The description of the protein encoded in this ORF:
Mouse L1 ORF2p, 150kDa, reverse transcriptase and endonuclease, essential for retrotransposition. This protein product was always anticipated/studied only indirectly.
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):
  2943-6788
Remarks:
Comments from Wai-Lun.Li__at__UCHSC.edu:
1) In preparing for submission, I found 4 "mutations" in the parent clone in which many other clones are
derived from. Quite frankly, I have no idea what effect those mutations have. 2) The nucleic acid data
sequence is the full length L1 spastic sequence. 3) The IRESs position # below are the sequences of L1spa
inserted into the EcoRI/NcoI site of pRF.

The pRF plasmid from Dr. Peter Sarnow's lab contains defective EMCV IRES in the intercistronic region and
should not be confused with the pRF plasmid from Anne Willis lab (IRESite IDs #184 and #231) or actually with
any other labs pRF having same name because of renilla and firefly luciferase reporters while different in
structure. In this work Li et al. (2006) obtained the plasmid from Sarnow's lab with defective EMCV IRES.
In an attempt to block cap-dependent translation of capped T7 transcripts they cloned additional functional
or defective EMCV IRES in front of the first reporter gene (Fig. 5). It is uncertain whether the plasmids were
stable in structure or whether unexpected recombination affected any of the observed results (possible
presence of shorter, monocistronic RNAs was not studied).

A promoter activity was known from the 5'-UTR region from a region called TF-type element, consisting of
several so called 7.2 monomers, each being a promoter. Li et al. (2006), Fig. 3B put an effort to isolate
known, strong cryptic promoter from TrkB coding sequence and demonstrates on Northern blot (Fig. 2) that the
cryptic promoter activity of both -400 to -1 (5'-UTR) and -201 to -1 (IGR) regions is "weak" compared to that
of TrkB. By the way they confirmed the CrPV IRES as re-cloned by them has no promoter activity, nor had a
312nt fragment of LINE-1 3'-UTR. Somehow in contrast the values in Fig. 6, right column show that CrPV has
some residual promoter activity.

In direct transfection of ARCA capped (mMESSAGE mMACHINE® T7 ULTRA Kit, Ambion) RNAs 400-1 LINE-1 IRES was
about 5x above the empty vector whereas 200-1 IGR LINE-1 IRES was 3x above the empty pRF vector (as well as
CrPV IGR IRES; although it is unclear from the article whether the 5'-UTR or IGR CrPV IRES was tested, this
information was provided additionally by Patrick Wai-Lun Li).

After thorough deletion analyses of both putative IRES regions Li et al. (2006) came up with 4 areas with no
detectable promoter activity while having some IRES activity in vivo:
-302 to -202
-101 to -1
-44 to -1
-138 to -86 (IGR IRES, actually placed in ORF1)

Unfortunately no direct RNA transfection data are available for those finally found regions, only the initial
-400 to -1 and -201 to -1 regions were tested as mentioned previously. It is uncertain whether the observed
"IRES activity" is due to a ribosomal read-through in a plasmid or not.

It is unclear in which experiments the pRF plasmid with defective/functional EMCV was used.
It is unclear which cells were used for in vivo expression. Authors answered "ltk-" but no more details.
It is noteworthy that in Fig. 5 the ARCA-capped transcripts were not translated by the cap-dependent scanning
mechanism as can be seen in part B with the defective EMCV IRES probably physically blocking ribosome
movement.
Citations:
Martin S. L., Cruceanu M., Branciforte D., Wai-Lun Li P., Kwok S. C., Hodges R. S., Williams M. C. (2005) LINE-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein. J. Mol. Biol. 348(3):549-561
Basame S., Wai-lun Li P., Howard G., Branciforte D., Keller D., Martin S. L. (2006) Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition. J. Mol. Biol. 357(2):351-357
IRESs:
IRES:
Version: 1 Last change: 2008-05-13 20:01:20
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  LINE-1_ORF1_-101_to_-1
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1686-1786
Conclusion:
  probably_not_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
This region has no promoter activity while about 9x activity of an empty pRF vector in vivo (Fig. 6). No
direct RNA transfection data are available, no verification of intactness of expected dicistronic mRNAs.
Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
Dmitriev S. E., Andreev D. E., Terenin I. M., Olovnikov I. A., Prassolov V. S., Merrick W. C., Shatsky I. N. (2007) Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated. Mol. Cell. Biol. 27(13):4685-4697
IRES:
Version: 1 Last change: 2008-05-13 20:01:20
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  LINE-1_ORF1_-302_to_-202
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1485-1585
Conclusion:
  probably_not_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
This region has no promoter activity while about 21x activity of an empty pRF vector in vivo (Fig. 6). No
direct RNA transfection data are available, no verification of intactness of expected dicistronic mRNAs.
Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
Dmitriev S. E., Andreev D. E., Terenin I. M., Olovnikov I. A., Prassolov V. S., Merrick W. C., Shatsky I. N. (2007) Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated. Mol. Cell. Biol. 27(13):4685-4697
IRES:
Version: 1 Last change: 2008-05-13 20:01:20
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  LINE-1_ORF2_-138_to_-86
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  2805-2857
Conclusion:
  probably_not_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
This region has no promoter activity while about 16x activity of an empty pRF vector in vivo (Fig. 7). No
direct RNA transfection data are available, no verification of intactness of expected dicistronic mRNAs.
Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
Dmitriev S. E., Andreev D. E., Terenin I. M., Olovnikov I. A., Prassolov V. S., Merrick W. C., Shatsky I. N. (2007) Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated. Mol. Cell. Biol. 27(13):4685-4697
IRES:
Version: 1 Last change: 2008-05-13 20:01:20
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  LINE-1_ORF1_-44_to_-1
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1740-1786
Conclusion:
  probably_not_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
This region has no promoter activity while about 5x activity of an empty pRF vector in vivo (Fig. 6). No
direct RNA transfection data are available, no verification of intactness of expected dicistronic mRNAs.
Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
Dmitriev S. E., Andreev D. E., Terenin I. M., Olovnikov I. A., Prassolov V. S., Merrick W. C., Shatsky I. N. (2007) Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5' untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated. Mol. Cell. Biol. 27(13):4685-4697
Last change to the database: 2015-04-16 16:45:23 GMT+1