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 description of the protein encoded in this ORF: cyclin D1
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): 210-1097
Remarks:
It seems the sequence NM_053056.2 is exactly same in the 5'-UTR as the sequence published in the Supplementary
data. However, it is not sure that the RT-PCR-amplified and cloned sequence was exactly same.
From several mRNA hits and especially based on the hit to a cDNA prepared by oligo-capping method (AK313136)
it seems the 5'-UTR is usually shorter, in this case by 51bp. Anyway, it seems the annotation was based on
this record:
LOCUS Z29078 4585 bp DNA linear PRI 05-MAY-1994
DEFINITION H.sapiens cyclin D1 gene promoter region.
ACCESSION Z29078
VERSION Z29078.1 GI:483600
[cut]
COMMENT On May 8, 1994 this sequence version replaced gi:469515.
FEATURES Location/Qualifiers
source 1..4585
/organism="Homo sapiens"
/mol_type="genomic DNA"
/db_xref="taxon:9606"
/clone="cyclin D1"
/cell_line="WI-38"
/cell_type="Fibroblast"
promoter <1..3015
prim_transcript 2970..>4585
exon 3016..3377
CDS 3179..>3377
/codon_start=1
/product="cyclin D1"
/protein_id="CAA82318.1"
/db_xref="GI:483601"
/db_xref="UniProtKB/TrEMBL:Q14092"
/translation="MEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSY
FKCVQKEVLRPCGRSSPPGCWR"
intron 3378..>4585
[cut]
And here is the alignment of the RefSeq sequence IRESite is using explaining why is the 5'-UTR so long:
>emb|Z29078.1| Gene info H.sapiens cyclin D1 gene promoter region
Length=4585
GENE ID: 595 CCND1 | cyclin D1 [Homo sapiens] (Over 100 PubMed links)
Score = 749 bits (405), Expect = 0.0
Identities = 408/409 (99%), Gaps = 1/409 (0%)
Strand=Plus/Plus
Query 1 CACACGGACTACAGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGGAGCCTCCAGAGGGCTG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 2970 CACACGGACTACAGGGGAGTTTTGTTGAAGTTGCAAAGTCCTGGAGCCTCCAGAGGGCTG 3029
Query 61 TCGGCGCAGTAGCAGCGAGCAGCAGAGTCCGCACGCTCCGGCGAGGGGCAGAAGAGCGCG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3030 TCGGCGCAGTAGCAGCGAGCAGCAGAGTCCGCACGCTCCGGCGAGGGGCAGAAGAGCGCG 3089
Query 121 AGGGAGCGCGGGGCAGCAGAAGCGAGAGCCGAGCGCGGACCCAGCCAGGACCCACAGCCC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3090 AGGGAGCGCGGGGCAGCAGAAGCGAGAGCCGAGCGCGGACCCAGCCAGGACCCACAGCCC 3149
Query 181 TCCCCAGCTGCCCAGGAAGAGCCCCAGCCATGGAACACCAGCTCCTGTGCTGCGAAGTGG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3150 TCCCCAGCTGCCCAGGAAGAGCCCCAGCCATGGAACACCAGCTCCTGTGCTGCGAAGTGG 3209
Query 241 AAACCATCCGCCGCGCGTACCCCGATGCCAACCTCCTCAACGACCGGGTGCTGCGGGCCA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3210 AAACCATCCGCCGCGCGTACCCCGATGCCAACCTCCTCAACGACCGGGTGCTGCGGGCCA 3269
Query 301 TGCTGAAGGCGGAGGAGACCTGCGCGCCCTCGGTGTCCTACTTCAAATGTGTGCAGAAGG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 3270 TGCTGAAGGCGGAGGAGACCTGCGCGCCCTCGGTGTCCTACTTCAAATGTGTGCAGAAGG 3329
Query 361 AGGTCCTGCCGTCCATGCGGAAGATCGTCGCCACCTGGATGCTGGAGGT 409
||||||| |||||||||||||||||||||||||||||||||||||||||
Sbjct 3330 AGGTCCT-CCGTCCATGCGGAAGATCGTCGCCACCTGGATGCTGGAGGT 3377
Regretfully, the experiments did not exclude the possibility of aberrantly spliced bicistronic mRNAs in the
cells. p27kip "IRES" was used as the positive control, known today to contain promoter. However, from the
different behavior of both CCND1 and c-myc IRESs from p27kip one could believe there is IRES in the former
two. Also increased presence of CCND1 and c-myc in polysomal fractions under low AKT activity cellular
conditions seems to confirm the presence of IRES.