The nucleic acid data:
IRESite Id: 124 Version: 9
Originaly submitted by: Blanka Vicenová
Reviewed by: Martin Mokrejš Last change: 2007-09-09 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:
  Kcna4
The genetic origin of this natural mRNA/+RNA:
  nuclear
The GenBankId GI:# number of the most similar mRNA/+RNA sequence to this one.
142358254 
Synonyms of the gene name:
Synonym: Kv1.4
Synonym: MGC124445
The mRNA/+RNA description: 
Mus musculus potassium voltage-gated channel, shaker-related subfamily, member 4 (Kcna4), mRNA.
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:
Mus musculus
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:
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:
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:
Homo sapiens HeLa (ATCC CCL-2)
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 2 Last change: 2007-09-06 00:00:00
Originaly submitted by: Blanka Vicenová Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
Kcna4
The description of the protein encoded in this ORF:
potassium voltage gated channel, shaker related subfamily, member 4
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):
  1200-3164
Remarks:
Mouse Kv1.4 gene generates at least three alternatively spliced transcripts 2.4, 3.5 and 4.5 kb in size
from a single promoter. The 2.4 kb transcript has been found only in skeletal muscle and in the C2C12
mouse myoblast cell line. The 3.5 kb and 4.5 kb transcripts are expressed in heart and brain, begin at a
single transcription initiation site and differ only by their use of alternative polyadenylation sites in
their 3'-UTRs.

The 5'-UTR of Kv1.4 is relatively long (1.2 kb), contains 18 upstream AUG codons and has a predicted complex
secondary structure with numerous stable hairpin structures. Several of the upstream AUG codons are contained
within a favorable context for initiation and are followed by open reading frames as long as 40 amino acids,
none of which is contiguous with the authentic coding region.

Negulescu et al. (1998) studied whole (1.2-kb) Kcna4 5'-UTR and also last 200b of the 5'-UTR which
shows activity comparable with that of the 1.2-kb fragment, suggesting that IRES activity is largely contained
within this region (which was not confirmed in Jang. et al. (2004) who utilized pRSTF based bicistronic
plasmids instead of SV40-CAT-LUV with impaired EMCV). Negulescu et al. (1998) did not use positive control.

The IRESite record is based on sequence pRst1.2F provided by J. Jimenez from the same lab where the Kv1.4
0.2kb IRES can be matched as well.

The 1.2kb IRES sequence provided here reasonably matches the original one from Negulescu et al. (1998)
shown Fig. 1A (1.2kb IRES starts two bases left from the arrow and further downstream so the sequence
12-AAGACTGGG...CCACCACCATG-1212 and is actually 1201bp long).

The sequence of Kv1.4 0.2 IRES annotated here is the XbaI - NcoI fragment and corresponds in Negulescu et
al. (1998) Fig. 1A to 979-TCTAGACGAGGCAAGCT...AAACCACCACCATG-1212.

Jang et al. (2004) have shown using direct RNA transcfection that only 1.2kb fragment of the 5'-UTR has IRES
activity (maybe also 1.0kb fragment, see Fig. 2B) but that the 0.2kb fragment does not have IRES activity.
Remarkably, even the 1.2kb fragment has only 9% activity of the positive control (CVB3). Aberrant splicing
of the plasmid with Kv1.4 was suspected by Kozak (2001) and shown later by Jang et al. (2004) in Fig. 1D and
by Jimenez et al. (2005) in Fig. 2C.

Note: Negulescu et al. (1998) reported they have deposited their sequences in GenBank under accession numbers
U03722 and U03723, but these sequences differ from the sequence in Fig. 1A in their article.
The sequence from Jang et al. (2004) Fig. 5A does only partially match the sequence shown in Negulescu
et al., (1998) in Fig. 1A: first 10 bases and the last 15 bases from Fig. 5A do not correspond to the
sequence in Fig. 1A.
Citations:
Negulescu D., Leong L. E., Chandy K. G., Semler B. L., Gutman G. A. (1998) Translation initiation of a cardiac voltage-gated potassium channel by internal ribosome entry. J. Biol. Chem. 273(32):20109-20113
Jang G. M., Leong L. E., Hoang L. T., Wang P. H., Gutman G. A., Semler B. L. (2004) Structurally distinct elements mediate internal ribosome entry within the 5'-noncoding region of a voltage-gated potassium channel mRNA. J. Biol. Chem. 279(46):47419-47430
Kozak M. (2001) New ways of initiating translation in eukaryotes?. Mol. Cell. Biol. 21(6):1899-1907
IRESs:
IRES:
Version: 7 Last change: 2007-09-06 00:00:00
Originaly submitted by: Blanka Vicenová Reviewed by: Martin Mokrejš
The IRES name:
  Kv1.4_1.2
Warning: please make ires_name same as the gene_name and optionally append to it coordinates. E.g. when gene/virus name is EMCV-R use EMCV-R_-222_to_-1 or EMCV-R_1-456, etc. but not Emcv-R-... or EMCV-222_to_-1. Please keep case of letters as well. This rewards when searching through the database.
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  3-1199
Conclusion:
  weakly_supported_IRES
How IRES boundaries were determined:
experimentally_determined
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
When the 1.2 kb Kv1.4 5'-UTR was examined for IRES activity after being inserted between the renilla and
firefly luciferase genes and expressed in HeLa cells from a dicistronic plasmid, smaller RNA species ~2.0 kb,
~2.2 kb and ~3.0 kb were detected using Northern blot in addition to full-length dicistronic RNAs. Their
presence suggests the involvement of RNA splicing or cryptic promoter presence. The possible presence of a
cryptic promoter in the Kv1.4 5'-UTR was ruled out using the promoter-less construct containing renilla and
firefly luciferase reporter genes and therefore it seems only aberrant splicing occurs.

To eliminate complications with alternatively spliced products Jang et al. (2004) transcribed in
vitro (possibly T7, uncapped, non-polyA tailed transcripts) although they did not state that clearly in
the article) dicistronic mRNAs and transfected them directly into HeLa cells. 5 and 15 hrs post-transfection,
smaller RNA species in cells were not detected anymore using Northern blot (Fig. 2C) indicating that
dicistronic RNAs are not subject to specific cleavage although one could object the method is not sufficiently
sensitive. Anyway, the IRES activity was 9.1% of the positive control CVB3 IRES.

Consistent with the previous finding that aberrant transcripts occur with Kv1.4 IRES inserted into DNA
plasmids are the findings of Jimenez et al. 2005 who have shown on Northern blot in Fig. 2C that
smaller transcripts can be found in transiently transfected cells. Further, they found Kv1.4 1.2kb IRES to be
not functional: 1.44% of the positive control CVB3 and also LEF-1 activity was 6.16% of the same control in
direct mRNA transfection (Fig. 4A and B).
Citations:
Negulescu D., Leong L. E., Chandy K. G., Semler B. L., Gutman G. A. (1998) Translation initiation of a cardiac voltage-gated potassium channel by internal ribosome entry. J. Biol. Chem. 273(32):20109-20113
Jang G. M., Leong L. E., Hoang L. T., Wang P. H., Gutman G. A., Semler B. L. (2004) Structurally distinct elements mediate internal ribosome entry within the 5'-noncoding region of a voltage-gated potassium channel mRNA. J. Biol. Chem. 279(46):47419-47430
Last change to the database: 2015-04-16 16:45:23 GMT+1