The nucleic acid data:
IRESite Id: 40 Version: 14
Originaly submitted by: Tomáš Mašek Submission date: 2005-06-26 00:00:00
Reviewed by: Martin Pospíšek Last change: 2009-08-29 19:20:40
IRESite record type:
  natural_transcript
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  end-to-end_full-length_mRNA
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule:
  CrPV
The genetic origin of this natural mRNA/+RNA:
  viral
The GenBankId GI:# number of exactly this mRNA/+RNA sequence:
21321708
The mRNA/+RNA description: 
Cricket paralysis virus (Dicistroviridae) nonstructural polyprotein and structural polyprotein genes, complete
genome.
The mRNA/+RNA sequence represented in the +DNA notation:


Credibility of mRNA sequence:
  end-to-end_sequence_completely_same_as_in_the_experiment
The organism containing this mRNA with IRES segment in its genome:
Cricket paralysis virus
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:
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:
homogeneous_population_of_molecules_confirmed
The organism used:
Drosophila melanogaster SL2
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:
Mus musculus
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 2 Last change: 2006-01-13 00:00:00
Originaly submitted by: Tomáš Mašek Reviewed by: Martin Pospíšek
The abbreviated name of this ORF/gene:
CrPVgp1
The description of the protein encoded in this ORF:
nonstructural 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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  709-6024
OPTIONAL: The function of the encoded protein
nonstructural polyprotein contains sequence motifs for RNA helicase, 3C-like proteinase and RNA-dependent RNA
polymerase
ORF
ORF position:   2
Version: 1
Originaly submitted by: Tomáš Mašek Reviewed by: Martin Pospíšek
The abbreviated name of this ORF/gene:
CrPVgp2
The description of the protein encoded in this ORF:
structural 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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  6214-8904
Remarks:
Translation of the second cistron is modulated by IGR (intragenic region) long 192 bp (6025-6216). Regions
1-708 and 6025-6232 were tested in two different bicistronic constructs for IRES activity. The IGR putative
IGR region was also point mutated and revealed that CCU start codon is used for the second ORF (candidate
sites were suggested by N-terminal protein "sequencing" showing Ala as the first aminoacid).


Dicistronic mRNA transcripts from pRF based plasmid were tested by direct RNA transfection of Drosophila SL2
cells. T7 in vitro transcripts were tested in rabbit reticulocyte lysates and both IRESs were functional. In
wheat germ extract only CrPV IGR IRES was functional (5'-NCR IRES not) while no positive control was shown. An
inverted repeat which includes the CCU codon 6187-6191:6212-6216 was confirmed by biochemical studies and
utilization of compensatory mutations.

Wilson et al. (2000) do not show any positive control IRES used in the intercistronic region except the
defective EMCV IRES (Figs. 3 and 7). The functional EMCV and defective EMCV IRESs were used in front of the
first cistron to affect cap-dependent translation (Figs. 5 and 6). From similar luciferase activities of DNA
(Fig. 3) versus RNA (Fig. 4) transfected cells one can conclude that there is no promoter activity
contributing the observed IRES activity.

From the work of Li et al. (2006), Fig. 3B it can be concluded there is no cryptic promoter in CrPV IRES,
although it is not clear into what plasmid context they re-cloned the CrPV IRES sequence and in which cells
they performed the test. Moreover, from direct transfection of ARCA capped (mMESSAGE mMACHINE® T7 ULTRA Kit,
Ambion) RNA it can be seen (Fig. 4) that CrPV IRES is about 3x above the negative control (empty pRF vector).
Unfortunately it is not clear which CrPV IRES was tested and whether the pRF plasmid contained in that moment
the defective EMCV IRES or not.
Citations:
Wilson J. E., Powell M. J., Hoover S. E., Sarnow P. (2000) Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites. Mol. Cell. Biol. 20(14):4990-4999
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
IRESs:
IRES:
Version: 4 Last change: 2009-09-13 22:14:20
Originaly submitted by: Tomáš Mašek Reviewed by: Martin Pospíšek
The IRES name:
  CrPV_5NCR
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1-708
Conclusion:
  putative_IRES
How IRES boundaries were determined:
unknown
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Wu et al. (2007) reported that their 5NTR IRES (247bp) was not functional in baculovirus-infected Sf21 cells.
Citations:
Wilson J. E., Powell M. J., Hoover S. E., Sarnow P. (2000) Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites. Mol. Cell. Biol. 20(14):4990-4999
Wu TY, Wu CY, Chen YJ, Chen CY, Wang CH (2007) The 5' untranslated region of Perina nuda virus (PnV) possesses a strong internal translation activity in baculovirus-infected insect cells. FEBS Lett. 581(16):3120-3126
IRES:
Version: 11 Last change: 2009-09-13 22:14:20
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  CrPV_IGR
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  6025-6216
Conclusion:
  strongly_supported_IRES
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  1
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  192
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -189
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  2
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Entire intergenic region of CrPV (6025-6213) and 3 bases of CrPVgp2. Inverted-repeat element, which is
predicted for bases 6187-6191 and 6212-6216 and in which is included initiator CCU codon, is necessary for
IRES activity.


Masoumi et al. (2003) tested IRES activity (Figure 2) and effect on viral yields of both NCR and IGR IRESs in
several insect cell lines using the original pRF plasmids containing the defective EMCV IRES. Only in one
cell line there were high yields of the FLuc protein.
Citations:
Wilson J. E., Powell M. J., Hoover S. E., Sarnow P. (2000) Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites. Mol. Cell. Biol. 20(14):4990-4999
Masoumi A, Hanzlik TN, Christian PD (2003) Functionality of the 5'- and intergenic IRES elements of cricket paralysis virus in a range of insect cell lines, and its relationship with viral activities. Virus Res. 2(94):113-120
IRES trans-acting factor (ITAFS):
IRES trans-acting factor (ITAF):
Version: 1 Last change: 2008-07-15 16:41:33
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
Type of the interaction between ITAF and the RNA subject to translation:
unknown
ITAF protein characteristics:
Version: 1 Last change: 2009-08-18 16:32:07
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
ITAF abbreviated name:
eIF4G
ITAF fullname:
eukaryotic translation initiation factor 4G
ITAF description (long):
eIF4G is the scaffolding protein bridging together the cap-binding protein eIF4E with other proteins of the initiating ribosome
3.1.2. Organisms or in vitro systems where this ITAF was functionally studied:
Organism or in vitro system where ITAF was shown:
Necessity of ITAF for translation in this particular organism or system:
no_effect_on_translation
Method used to demonstrate ITAF effect:
in_vivo
The organism where action of this ITAF was studied:
Saccharomyces cerevisiae
Remarks:
Depletion of eIF4G to 37% reduced CrPV IGR IRES to 75%. Further, over-expression of eIF4G does not increase
CrPV IRES activity. Data from Figure 2A,B.
Citations:
Gilbert W. V., Zhou K., Butler T. K., Doudna J. A. (2007) Cap-independent translation is required for starvation-induced differentiation in yeast. Science. 317(5842):1224-1227
Regions with experimentally determined secondary structures:
A region with the experimentally determined secondary structure:
IRESite 2D Struct Id: 44
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The function of the 2D structure:
IRES
The 2D structure causes frameshift:
unknown
The absolute position of the experimentally mapped region (the range includes START and STOP codons or their equivalents):
6030-6219
The underlying nucleic acid sequence and structure of the mapped region:



Remarks:
The pseudoknot interactions were omitted from the CryoEM structure (PDB:2NOQ).
Citations:
Schüler M, Connell SR, Lescoute A, Giesebrecht J, Dabrowski M, Schroeer B, Mielke T, Penczek PA, Westhof E, Spahn CM (2006) Structure of the ribosome-bound cricket paralysis virus IRES RNA. Nat. Struct. Mol. Biol. 12(13):1092-1096
Last change to the database: 2015-04-16 16:45:23 GMT+1