IRESite_Id:124 summary record describing characteristics of Kv1.4

IRESite: The database of experimentally verified IRES structures

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:

AAGACTGGGA TGGATACTGG AGAAGGAATG CAGGCTTAAC AAGTGATCGC TGCTGTCTAG GATTTTGAGT CTTTTTCGGA GAACCTTGAC TTCCGTTCCC 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

AGCCCATGTC TGCTGTGCCG AACTCCAGAG GAACCAGAAA TCTCCGGGGT CTACCTTGGG GCGTCCCCAA TCTCCACCTC TGGGCTCCAG TAACGAGGAC 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

TCTGCAATAC CCCCTAGCCC CCTGGCCAAG ACAACCGAAC TTGTTCCGTG GATATTTGGG ATCCTCCACC TGCCAAACCT GAGCGATTTT TTTTGTACTG 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

CGCCCCCACC CCCAATGATT CTGCCCCTCC TCCAGCTGTT GCAGCGTGGA AAAGGGGAAA CAAATCACCG GGGGGGATTT TTTTCGTCTA TTTTTATTTT 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

TCGCACTTGC TGGGAATGGT GAAGTGCTTC TTGTGAATGA TTCTAGCCAA AGGATGCTCT TCATTTCCTG CTTTCTATGG AGACCTCAGT GTTGAGTTTG 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

CCTCTGCTGG AACTGCGTCT ACCACCTTCT CTACCTTCCA AGGTCTTTGC CTCTATCTAC AACCTGGCAT TGTCTGTGGG TCCATGAAGG CTTGGATGAC 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

CTTAGAGGGA AGGTCTGGGA GTCCACCCTC ATAGACTAAG CAGCAATGGC TGGGCATATT TTAAGCCGCA TTTTAACATG GGTCAAGCCA TCAGTAGAAG 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

GCAAGTGCTA AGACTAAAGA CTTATTTGAA TTTTATTTAA ATTAGATGGA CTGGGCCTTG GCCAATTTCC ATGCAAGAAA AAGTATATTT CATTTTCTAG 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

GCACAACTTC TGAGTGTCAG ATACTTGCTG TCTTTGAGTC TTGTGGCGTC ATCACCGGAC AGCATCCCAG ACAGACTTCC AGATTTGAAC ATCTACCCCC 
^801       ^811       ^821       ^831       ^841       ^851       ^861       ^871       ^881       ^891       

CAACACGTAG GTGTATGGGA GACCACATCA TTTCATGACT TATGTTTGAG GAACACTAGG CTGTTGTCTA GACGAGGCAA GCTCTGGAAA GCAACGCCGA 
^901       ^911       ^921       ^931       ^941       ^951       ^961       ^971       ^981       ^991       

GTCTCTGAGA AGAGGGAGCA TAGGCTGTGC TGATTTAAAA ACAGAAAATG CAAAGTTGGA CTGAAAATAT CCCACGTCTT CTAAGCAATC TGCTTAAGGC 
^1001      ^1011      ^1021      ^1031      ^1041      ^1051      ^1061      ^1071      ^1081      ^1091      

TTCCAAACTT ACCTTAATTT GGTAAGAAAA TAAGCTGCCC TATTTTTCTT TCTTCTTCTC TTACAACTGG AAGCAGCCAT TTCCCCAAAC CACCACCATG 
^1101      ^1111      ^1121      ^1131      ^1141      ^1151      ^1161      ^1171      ^1181      ^1191      

GAAGTGGCGA TGGTGAGTGC CGAGAGCTCA GGGTGCAACA GCCATATGCC TTATGGTTAT GCAGCCCAGG CCAGGGCCCG AGAGCGGGAG AGACTCGCTC 
^1201      ^1211      ^1221      ^1231      ^1241      ^1251      ^1261      ^1271      ^1281      ^1291      

ACTCCAGGGC AGCTGCAGCT GCTGCTGTCG CAGCTGCCAC GGCTGCTGTC GAAGGCACTG GGGGTTCTGG TGGAGGCCCC CACCACCATC ATCAGACACG 
^1301      ^1311      ^1321      ^1331      ^1341      ^1351      ^1361      ^1371      ^1381      ^1391      

TGGGGCCTAC TCCTCCCATG ATCCTCAAGG TAGCCGTGGA AGTAGAAGGA GGAGGCGTCA GCGAACTGAG AAGAAGAAAC TCCACCACAG GCAGAGCAGT 
^1401      ^1411      ^1421      ^1431      ^1441      ^1451      ^1461      ^1471      ^1481      ^1491      

TTTCCTCATT GCTCAGACCT GATGCCCAGT GGCTCTGAAG AGAAGATCCT GAGGGAGCTA AGCGAGGAAG AGGAAGACGA GGAGGAGGAA GAGGAGGAGG 
^1501      ^1511      ^1521      ^1531      ^1541      ^1551      ^1561      ^1571      ^1581      ^1591      

AGGAGGAGGG AAGGTTTTAC TATAGTGAAG AAGACCATGG GGATGGGTGT TCGTACACAG ACCTGCTGCC ACAGGATGAT GGGGGTGGTG GCGGCTACAG 
^1601      ^1611      ^1621      ^1631      ^1641      ^1651      ^1661      ^1671      ^1681      ^1691      

TTCAGTCCGC TATAGTGACT GCTGTGAACG TGTGGTGATA AATGTGTCTG GTCTACGCTT CGAAACCCAA ATGAAAACTT TGGCCCAGTT TCCAGAAACT 
^1701      ^1711      ^1721      ^1731      ^1741      ^1751      ^1761      ^1771      ^1781      ^1791      

CTGTTGGGAG ACCCTGAGAA GAGGACTCAG TACTTCGACC CTTTGCGCAA TGAGTATTTT TTTGATCGGA ACCGACCCAG CTTTGATGCC ATTTTGTATT 
^1801      ^1811      ^1821      ^1831      ^1841      ^1851      ^1861      ^1871      ^1881      ^1891      

ATTACCAGTC AGGAGGCCGC CTGAAGAGAC CAGTCAATGT CCCCTTTGAT ATCTTCACCG AGGAGGTGAA GTTCTATCAG TTGGGAGAGG AAGCCCTGCT 
^1901      ^1911      ^1921      ^1931      ^1941      ^1951      ^1961      ^1971      ^1981      ^1991      

CAAGTTCCGG GAGGATGAGG GCTTTGTGAG AGAAGAGGAG GACAGGGCTC TGCCAGAAAA TGAATTTAAA AAACAGATTT GGCTTCTCTT TGAATATCCA 
^2001      ^2011      ^2021      ^2031      ^2041      ^2051      ^2061      ^2071      ^2081      ^2091      

GAGAGTTCTA GCCCTGCCAG GGGTATAGCC ATTGTGTCTG TCCTGGTCAT CTTAATCTCT ATTGTCATAT TTTGCCTGGA AACCTTGCCG GAGTTCAGGG 
^2101      ^2111      ^2121      ^2131      ^2141      ^2151      ^2161      ^2171      ^2181      ^2191      

ATGATAGGGA CCTTATTATG GCCCTCAGTG CAGGCGGGCA CAGCAGATTG CTGAATGACA CCTCGGCACC CCACCTGGAG AACTCAGGGC ACACAATATT 
^2201      ^2211      ^2221      ^2231      ^2241      ^2251      ^2261      ^2271      ^2281      ^2291      

CAATGACCCT TTCTTCATCG TGGAGACAGT GTGTATTGTG TGGTTTTCCT TTGAGTTTGT GGTTCGATGC TTTGCTTGTC CCAGCCAAGC ACTCTTCTTC 
^2301      ^2311      ^2321      ^2331      ^2341      ^2351      ^2361      ^2371      ^2381      ^2391      

AAAAACATCA TGAACATCAT TGATATCGTC TCCATTTTGC CTTACTTCAT CACTCTGGGC ACTGACCTGG CCCAACAGCA GGGGGGTGGC AATGGCCAGC 
^2401      ^2411      ^2421      ^2431      ^2441      ^2451      ^2461      ^2471      ^2481      ^2491      

AGCAGCAGGC CATGTCCTTT GCCATCCTTA GGATCATTCG TCTGGTCCGA GTATTCCGGA TCTTCAAGCT CTCCAGACAC TCCAAAGGCC TGCAGATCCT 
^2501      ^2511      ^2521      ^2531      ^2541      ^2551      ^2561      ^2571      ^2581      ^2591      

GGGCCACACC CTAAGAGCCA GCATGCGGGA ACTGGGCCTT CTTATCTTTT TCCTCTTCAT CGGGGTTATC CTCTTTTCCA GCGCTGTGTA TTTTGCAGAG 
^2601      ^2611      ^2621      ^2631      ^2641      ^2651      ^2661      ^2671      ^2681      ^2691      

GCGGATGAAC CCACTACCCA TTTCCAAAGC ATTCCAGATG CGTTTTGGTG GGCTGTGGTA ACCATGACAA CTGTGGGCTA TGGGGACATG AAGCCCATCA 
^2701      ^2711      ^2721      ^2731      ^2741      ^2751      ^2761      ^2771      ^2781      ^2791      

CAGTCGGGGG AAAGATTGTG GGGTCCCTGT GTGCCATTGC GGGTGTCTTA ACCATTGCTT TGCCTGTGCC GGTGATTGTG TCTAACTTTA ACTATTTCTA 
^2801      ^2811      ^2821      ^2831      ^2841      ^2851      ^2861      ^2871      ^2881      ^2891      

CCACAGAGAG ACTGAAAATG AAGAACAGAC CCAGCTGACA CAAAACGCAG TCAGTTGTCC ATACCTACCT TCTAATTTGC TCAAGAAATT TCGGAGCTCC 
^2901      ^2911      ^2921      ^2931      ^2941      ^2951      ^2961      ^2971      ^2981      ^2991      

ACTTCTTCTT CCCTGGGGGA CAAGTCAGAG TATCTAGAGA TGGAAGAAGG GGTCAAGGAA TCATTATGTG GAAAGGAGGA GAAGTGTCAG GGAAAGGGAG 
^3001      ^3011      ^3021      ^3031      ^3041      ^3051      ^3061      ^3071      ^3081      ^3091      

ATGAGAGCGA GACAGATAAA AACAACTGTT CTAATGCAAA GGCTGTGGAG ACTGATGTGT GAATTGTTCT CTCCACCTGC CACTGTCCCC CCATCTCCAA 
^3101      ^3111      ^3121      ^3131      ^3141      ^3151      ^3161      ^3171      ^3181      ^3191      

ATATATTCAT ACATAGAGAA TGCAGTTATG AAAATGAGAT ATGCAAACGA TTGCACTGCA TACAGTGATA TGCTGTTTAA TGGTAATACA TGGCATAATT 
^3201      ^3211      ^3221      ^3231      ^3241      ^3251      ^3261      ^3271      ^3281      ^3291      

GTGACTAAAC GTGTATTGCA TATCAAATAA ATGATACATC TTGGAGAAGA GGGAGGCATT AAAAACAGCA GATCTATCTT TATATTTTTT AATAGAATGC 
^3301      ^3311      ^3321      ^3331      ^3341      ^3351      ^3361      ^3371      ^3381      ^3391      

AAGAATTTTG CACATAATGG GAAAAATGTT AATAGTAAAG GTGGTTCTGA GGAGAGTGAG TGTGTGTGTG TGAGAGAGCG AGAGAGTGTG TGTACCTGGG 
^3401      ^3411      ^3421      ^3431      ^3441      ^3451      ^3461      ^3471      ^3481      ^3491      

TATGTAAGTA AATTGTCAAC ACTGTTGGGA ATTGTGCCGT GATGGAAAAA GTTGGCATTC TGAAGTATTT ACTATGTAAG AACTAATGAA TTTGAGCAGT 
^3501      ^3511      ^3521      ^3531      ^3541      ^3551      ^3561      ^3571      ^3581      ^3591      

CTTTTACCAG TGTTTTAATA ACATCTCCTA TGTCTTTGGA TTCTGTAGTT GTTTTTTAGA AATTATAAGA ATTACTGTGT AGAAAAAAGA GAAAGTAAAT 
^3601      ^3611      ^3621      ^3631      ^3641      ^3651      ^3661      ^3671      ^3681      ^3691      

TATTTAATAG AATATAGGTC ACAATTTAAT CTTGGATTTA ATTAAAGTTT ATTTTTAACT GGAAATTAAC TTTTGAAAAG GCTGCAGGGG CCTTTAGAAA 
^3701      ^3711      ^3721      ^3731      ^3741      ^3751      ^3761      ^3771      ^3781      ^3791      

TTGATTATAT TTTATTATTA ATTTTGGGGA GATATAATAG CAAATGCCTA ACATTCTGGA GGAAATGTAA CAAGTTTTGT TCACAGGTCT TAAAACTGGA 
^3801      ^3811      ^3821      ^3831      ^3841      ^3851      ^3861      ^3871      ^3881      ^3891      

TTTTTTTTTC TTTTGCACTA CTTTCTATGC CGAAGCCCGA GAGAGACTTC ATACTATGAA TGTTTACTAA TGCACCAATC AGTTCAATGA CAATCATTGG 
^3901      ^3911      ^3921      ^3931      ^3941      ^3951      ^3961      ^3971      ^3981      ^3991      

AAGAATGGGT TCTTCGTCTC ATTTATTGTT CTCTTCCTTT CGTGAGACTA ATGGCCACAC AAATAACAGC ACATGATTCC TGCTTTAAAA TCCGAACAAC 
^4001      ^4011      ^4021      ^4031      ^4041      ^4051      ^4061      ^4071      ^4081      ^4091      

TCATCTACAA AGGGACTATG AAGTAACGTT CAGCAGCCGA ATCTTTTGAA ATTGGTTTGT TACGATGATG CTTCAGAAAC CATACTATTT TCAATACTCT 
^4101      ^4111      ^4121      ^4131      ^4141      ^4151      ^4161      ^4171      ^4181      ^4191      

TCTGCCTTTT AAGTCCAGAA TAATTTAACC AAAGTTATTG CATGCACAGA GAGAATTCTT GGAGAAATAA GGCACCCAGC AGCTACAGCA ATTATGGCTT 
^4201      ^4211      ^4221      ^4231      ^4241      ^4251      ^4261      ^4271      ^4281      ^4291      

AAGATCTTTT TATTAAATAT GCAACAAAAT GCTAGATTTA AGTCCCTTTT GTGTATGTGT GTGTGTGTCT GTGTGTGTCT GTGTGTGCAG CTGCAAAAAC 
^4301      ^4311      ^4321      ^4331      ^4341      ^4351      ^4361      ^4371      ^4381      ^4391      

AAAACAGAAC ACGGAATGTG GGGATTCGCC TCAAATGGTA AAGCACTCTG CTAAATCAGA GTTAGGGAAG AATGGTTTTA GAGCATTTGG TTTCCCAATG 
^4401      ^4411      ^4421      ^4431      ^4441      ^4451      ^4461      ^4471      ^4481      ^4491      

GTCATTGTAA ATCATTATCA TCTTTATCAC AGGGGCTTCC TGGGGATTAT ACTTCCATTT ACCTCACAAT AGCTATTCTT GAGTTTGGAG TAGGAACAAG 
^4501      ^4511      ^4521      ^4531      ^4541      ^4551      ^4561      ^4571      ^4581      ^4591      

GAATGCACCA CGAACAACTG TAGCACCATA AAATACAGCT CAGAACACTC AGTATAAACA CATAGGAGGC ATTACTAAAC AATTCATTAA AACAAAACAA 
^4601      ^4611      ^4621      ^4631      ^4641      ^4651      ^4661      ^4671      ^4681      ^4691      

AATCTTCTTT ATAAATGGTT GCCCTGAGCA TTGGGAAGAA ATCCCATGTC CACTATTTCA GTCCATAGCA AACGTGAGC
^4701      ^4711      ^4721      ^4731      ^4741      ^4751      ^4761      ^4771

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):

GACTGGGATG GATACTGGAG AAGGAATGCA GGCTTAACAA GTGATCGCTG CTGTCTAGGA TTTTGAGTCT TTTTCGGAGA ACCTTGACTT CCGTTCCCAG 
^3         ^13        ^23        ^33        ^43        ^53        ^63        ^73        ^83        ^93        

CCCATGTCTG CTGTGCCGAA CTCCAGAGGA ACCAGAAATC TCCGGGGTCT ACCTTGGGGC GTCCCCAATC TCCACCTCTG GGCTCCAGTA ACGAGGACTC 
^103       ^113       ^123       ^133       ^143       ^153       ^163       ^173       ^183       ^193       

TGCAATACCC CCTAGCCCCC TGGCCAAGAC AACCGAACTT GTTCCGTGGA TATTTGGGAT CCTCCACCTG CCAAACCTGA GCGATTTTTT TTGTACTGCG 
^203       ^213       ^223       ^233       ^243       ^253       ^263       ^273       ^283       ^293       

CCCCCACCCC CAATGATTCT GCCCCTCCTC CAGCTGTTGC AGCGTGGAAA AGGGGAAACA AATCACCGGG GGGGATTTTT TTCGTCTATT TTTATTTTTC 
^303       ^313       ^323       ^333       ^343       ^353       ^363       ^373       ^383       ^393       

GCACTTGCTG GGAATGGTGA AGTGCTTCTT GTGAATGATT CTAGCCAAAG GATGCTCTTC ATTTCCTGCT TTCTATGGAG ACCTCAGTGT TGAGTTTGCC 
^403       ^413       ^423       ^433       ^443       ^453       ^463       ^473       ^483       ^493       

TCTGCTGGAA CTGCGTCTAC CACCTTCTCT ACCTTCCAAG GTCTTTGCCT CTATCTACAA CCTGGCATTG TCTGTGGGTC CATGAAGGCT TGGATGACCT 
^503       ^513       ^523       ^533       ^543       ^553       ^563       ^573       ^583       ^593       

TAGAGGGAAG GTCTGGGAGT CCACCCTCAT AGACTAAGCA GCAATGGCTG GGCATATTTT AAGCCGCATT TTAACATGGG TCAAGCCATC AGTAGAAGGC 
^603       ^613       ^623       ^633       ^643       ^653       ^663       ^673       ^683       ^693       

AAGTGCTAAG ACTAAAGACT TATTTGAATT TTATTTAAAT TAGATGGACT GGGCCTTGGC CAATTTCCAT GCAAGAAAAA GTATATTTCA TTTTCTAGGC 
^703       ^713       ^723       ^733       ^743       ^753       ^763       ^773       ^783       ^793       

ACAACTTCTG AGTGTCAGAT ACTTGCTGTC TTTGAGTCTT GTGGCGTCAT CACCGGACAG CATCCCAGAC AGACTTCCAG ATTTGAACAT CTACCCCCCA 
^803       ^813       ^823       ^833       ^843       ^853       ^863       ^873       ^883       ^893       

ACACGTAGGT GTATGGGAGA CCACATCATT TCATGACTTA TGTTTGAGGA ACACTAGGCT GTTGTCTAGA CGAGGCAAGC TCTGGAAAGC AACGCCGAGT 
^903       ^913       ^923       ^933       ^943       ^953       ^963       ^973       ^983       ^993       

CTCTGAGAAG AGGGAGCATA GGCTGTGCTG ATTTAAAAAC AGAAAATGCA AAGTTGGACT GAAAATATCC CACGTCTTCT AAGCAATCTG CTTAAGGCTT 
^1003      ^1013      ^1023      ^1033      ^1043      ^1053      ^1063      ^1073      ^1083      ^1093      

CCAAACTTAC CTTAATTTGG TAAGAAAATA AGCTGCCCTA TTTTTCTTTC TTCTTCTCTT ACAACTGGAA GCAGCCATTT CCCCAAACCA CCACCAT
^1103      ^1113      ^1123      ^1133      ^1143      ^1153      ^1163      ^1173      ^1183      ^1193

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:

Last change to the database: 2019-03-18 09:32:49 GMT+1