IRESite_Id:106 summary record describing characteristics of BAG1

IRESite: The database of experimentally verified IRES structures

The nucleic acid data:

IRESite Id: `106`	Version: `10`
Originaly submitted by: Zuzana Feketová	Submission date: `2005-12-19 00:00:00`
Reviewed by: Martin Mokrejš	Last change: `2009-09-02 15:31:03`

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: `BAG-1`	The genetic origin of this natural mRNA/+RNA: `nuclear`

The GenBankId GI:# number of the most similar mRNA/+RNA sequence to this one.
124494250

Synonyms of the gene name:

Synonym: RAP46

Synonym: HAP46

Synonym: Bag1

The mRNA/+RNA description:

Homo sapiens BCL2-associated athanogene (BAG1), mRNA.

The mRNA/+RNA sequence represented in the +DNA notation:

gcaggccggg gcggggcTGG GAAGTAGTCG GGCGGGGTTG TGAGACGCCG CGCTCAGCTT CCATCGCTGG GCGGTCAACA AGTGCGGGCC TGGCTCAGCG 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

CGGGGGGGCG CGGAGACCGC GAGGCGACCG GGAGCGGCTG GGTTCCCGGC TGCGCGCCCT TCGGCCAGGC CGGGAGCCGC GCCAGTCGGA GCCCCCGGCC 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

CAGCGTGGTC CGCCTCCCTC TCGGCGTCCA CCTGCCCGGA GTACTGCCAG CGGGCATGAC CGACCCACCA GGGGCGCCGC CGCCGGCGCT CGCAGGCCGC 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

GGATGAAGAA GAAAACCCGG CGCCGCTCGA CCCGGAGCGA GGAGTTGACC CGGAGCGAGG AGTTGACCCT GAGTGAGGAA GCGACCTGGA GTGAAGAGGC 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

GACCCAGAGT GAGGAGGCGA CCCAGGGCGA AGAGATGAAT CGGAGCCAGG AGGTGACCCG GGACGAGGAG TCGACCCGGA GCGAGGAGGT GACCAGGGAG 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

GAAATGGCGG CAGCTGGGCT CACCGTGACT GTCACCCACA GCAATGAGAA GCACGACCTT CATGTTACCT CCCAGCAGGG CAGCAGTGAA CCAGTTGTCC 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

AAGACCTGGC CCAGGTTGTT GAAGAGGTCA TAGGGGTTCC ACAGTCTTTT CAGAAACTCA TATTTAAGGG AAAATCTCTG AAGGAAATGG AAACACCGTT 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

GTCAGCACTT GGAATACAAG ATGGTTGCCG GGTCATGTTA ATTGGGAAAA AGAACAGTCC ACAGGAAGAG GTTGAACTAA AGAAGTTGAA ACATTTGGAG 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

AAGTCTGTGG AGAAGATAGC TGACCAGCTG GAAGAGTTGA ATAAAGAGCT TACTGGAATC CAGCAGGGTT TTCTGCCCAA GGATTTGCAA GCTGAAGCTC 
^801       ^811       ^821       ^831       ^841       ^851       ^861       ^871       ^881       ^891       

TCTGCAAACT TGATAGGAGA GTAAAAGCCA CAATAGAGCA GTTTATGAAG ATCTTGGAGG AGATTGACAC ACTGATCCTG CCAGAAAATT TCAAAGACAG 
^901       ^911       ^921       ^931       ^941       ^951       ^961       ^971       ^981       ^991       

TAGATTGAAA AGGAAAGGCT TGGTAAAAAA GGTTCAGGCA TTCCTAGCCG AGTGTGACAC AGTGGAGCAG AACATCTGCC AGGAGACTGA GCGGCTGCAG 
^1001      ^1011      ^1021      ^1031      ^1041      ^1051      ^1061      ^1071      ^1081      ^1091      

TCTACAAACT TTGCCCTGGC CGAGTGAGGT GTAGCAGAAA AAGGCTGTGC TGCCCTGAAG AATGGCGCCA CCAGCTCTGC CGTCTCTGGA GCGGAATTTA 
^1101      ^1111      ^1121      ^1131      ^1141      ^1151      ^1161      ^1171      ^1181      ^1191      

CCTGATTTCT TCAGGGCTGC TGGGGGCAAC TGGCCATTTG CCAATTTTCC TACTCTCACA CTGGTTCTCA ATGAAAAATA GTGTCTTTGT GATTTTGAGT 
^1201      ^1211      ^1221      ^1231      ^1241      ^1251      ^1261      ^1271      ^1281      ^1291      

AAAGCTCCTA TCTGTTTT
^1301      ^1311

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:

Homo sapiens

A promoter reported in cDNA corresponding to IRES sequence:
no The total number of notable open-reading frames (ORFs):
4

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:
homogeneous_population_of_molecules_confirmed

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:
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: `3`	Last change: `2007-09-11 00:00:00`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Mokrejš

The abbreviated name of this ORF/gene:
Bag-1L

The description of the protein encoded in this ORF:
50 kDa isoform of Bag-1 initiated at a non-canonical CUG codon, contains SV40-like nuclear localization signal and is found mainly in nucleus.

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:

yes

The ORF absolute position (the base range includes START and STOP codons or their equivalents):

90-1127

ORF

ORF position:

2

Version: `2`	Last change: `2007-09-11 00:00:00`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Mokrejš

The abbreviated name of this ORF/gene:
BAG-1M

The description of the protein encoded in this ORF:
p46 isoform of BAG-1, which is distributed in nucleus and in cytoplasm.

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:

yes

The ORF absolute position (the base range includes START and STOP codons or their equivalents):

303-1127

ORF

ORF position:

3

Version: `2`	Last change: `2007-09-11 00:00:00`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Mokrejš

The abbreviated name of this ORF/gene:
BAG-1S

The description of the protein encoded in this ORF:
The most common p36 isoform of BAG-1, which predominantly cytoplasmic and is possibly translated by IRES mechanism.

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:

yes

The ORF absolute position (the base range includes START and STOP codons or their equivalents):

435-1127

ORF

ORF position:

4

Version: `1`	Last change: `2007-09-11 00:00:00`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Mokrejš

The abbreviated name of this ORF/gene:
BAG-1 rare p29 isoform

The description of the protein encoded in this ORF:
rare p29 isoform

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:

yes

The ORF absolute position (the base range includes START and STOP codons or their equivalents):

504-1127

Remarks:

It appears the RefSeq annotation of Bag1 mRNA (NM_004323.4) is an over-prediction. Several mRNAs in GenBank
confirm only the RefSeq model up to bases around position 1320.

CR618366
Z35491 - has poly(A)-tail, 1.3kb
BC001936 - has poly(A)-tail, 1.3kb
AF022224 - has poly(A)-tail, 1.29kb
AK222749 - clone prepared by oligo-capping method, 1.23kb sequenced, no poly(A)-tail
U46917 - no poly(A)-tail, 1.28kb
CR618567 - no poly(A)-tail, 1.22kb
CR603873 - no poly(A)-tail, 1.21kb

Based on the alignment to U46917 IRESite took over only the first 1318bp. The short extension found in 5'-UTR
of CR618366 which propagated into the RefSeq record is emphasized in lower-case letters. We cannot comment on
this more than that the extension is not conformed by other mRNA sequences.

All initiation codons of p50, p46, p36, p29 are in poor Kozak context and all are in-frame, so individual
isoforms of the BAG-1 protein share C-terminus and are shorter at the N-terminal end. The p29 isoform is
extremely rare and it is not always detected.

RNase protection method was used only to study integrity of pRBF in vivo transcripts (Fig. 3C).

Important data from several figures could not be processed due to the lack of sequences available to IRESite.
If the results are not an artifact they show that HRV IRES performs badly compared to c-myc IRES, EMCV IRES
and Bag-1 p36 IRES (pRBF). Figure 2B shows individual activities of renilla and firefly luciferases of pRF,
pRAF, pRBF, pREMCVF, pRHRVF, pRMF normalized to co-transfection control pcDNA3.1.

Figure 2C shows individual activities of renilla luciferase and chloramphenicol acetyltransferase of pRCAT,
pRBCAT, pRMCAT normalized to co-transfection control pcDNA3.1.

Figure 4 shows activity of IRES in various monocistronic constructs and relative amounts of various protein
isoforms produced in transiently transfected HeLa cells.

Citations:

Coldwell M. J., deSchoolmeester M. L., Fraser G. A., Pickering B. M., Packham G., Willis A. E. (2001) The p36 isoform of BAG-1 is translated by internal ribosome entry following heat shock. Oncogene. 20(30):4095-4100

IRESs:

IRES:

Version: `11`	Last change: `2008-05-12 13:53:41`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Mokrejš
The IRES name: `BAG1_p36delta236nt` 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):

303-432

Conclusion:
putative_IRES

How IRES boundaries were determined:
experimentally_determined

The sequence of IRES region aligned to its secondary structure (if available):

ATGAAGAAGA AAACCCGGCG CCGCTCGACC CGGAGCGAGG AGTTGACCCG GAGCGAGGAG TTGACCCTGA GTGAGGAAGC GACCTGGAGT GAAGAGGCGA 
.......... ....((..(( ......(((( (.......)) .)))....(( (..(((.... )))...))). .))....... .((((((..( (......)). 
^303       ^313       ^323       ^333       ^343       ^353       ^363       ^373       ^383       ^393       

CCCAGAGTGA GGAGGCGACC CAGGGCGAAG
.)))).)).. )).)))).)) ).)))))...
^403       ^413       ^423

There is no Vienna RNA package installed on the server or some error/warning messages were output. Due to that maybe we cannot prepare 2D structures for display. The error/warning message was:

..............((..((......(((((.......)).)))....(((..(((....)))...)))..))........((((((..((......))..)))).))..)).)))).))).)))))...
ERROR: unbalanced brackets in make_pair_table

STDOUT was:

Remarks:

This IRES (130nt, Fig. 5) was studied in pR3deltaF plasmid and retains 75% of the activity of p36 IRES (pRBF).
It is essential for the expression of Bag-1S during the recovery period of cells that have been stressed by
heat shock (Fig. 6).

Citations:

IRES:

Version: `7`	Last change: `2008-12-10 00:15:14`
Originaly submitted by: Martin Mokrejš	Reviewed by: Martin Mokrejš
The IRES name: `BAG1_p36` 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):

67-432

Conclusion:
putative_IRES

How IRES boundaries were determined:
experimentally_determined

The sequence of IRES region aligned to its secondary structure (if available):

CTGGGCGGTC AACAAGTGCG GGCCTGGCTC AGCGCGGGGG GGCGCGGAGA CCGCGAGGCG ACCGGGAGCG GCTGGGTTCC CGGCTGCGCG CCCTTCGGCC 
^67        ^77        ^87        ^97        ^107       ^117       ^127       ^137       ^147       ^157       

AGGCCGGGAG CCGCGCCAGT CGGAGCCCCC GGCCCAGCGT GGTCCGCCTC CCTCTCGGCG TCCACCTGCC CGGAGTACTG CCAGCGGGCA TGACCGACCC 
^167       ^177       ^187       ^197       ^207       ^217       ^227       ^237       ^247       ^257       

ACCAGGGGCG CCGCCGCCGG CGCTCGCAGG CCGCGGATGA AGAAGAAAAC CCGGCGCCGC TCGACCCGGA GCGAGGAGTT GACCCGGAGC GAGGAGTTGA 
^267       ^277       ^287       ^297       ^307       ^317       ^327       ^337       ^347       ^357       

CCCTGAGTGA GGAAGCGACC TGGAGTGAAG AGGCGACCCA GAGTGAGGAG GCGACCCAGG GCGAAG
^367       ^377       ^387       ^397       ^407       ^417       ^427

Remarks:

The longer, 366nt long IRES referred to as p36 IRES in the article and was cloned into pRBF plasmid.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 was not found to bind to BAG-1 IRES (Cammas et al., 2007)
which binds to HRV-2 and Apaf-1 IRESs.

Citations:

Cammas A., Pileur F., Bonnal S., Lewis S. M., Leveque N., Holcik M., Vagner S. (2007) Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs. Mol. Biol. Cell. 18(12):5048-5059

RNA:protein interactions:

The RNA:protein interaction:

Version: `7`	Last change: `2009-09-01 18:20:13`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Mokrejš

The description of the protein interacting with the RNA:
PTB /polypyrimidine tract binding protein/

The interacting RNA base range: 265-365

The function of the protein interaction:

Probably RNA chaperone, which enables to form the correct structure for 40S binding, probably also a part of

The organism where this RNA:protein interaction occurs:

Homo sapiens

Remarks:

The interaction was identified using EMSA method. The protein seems to bind several times within the 265-365
fragment and it even might form dimers.

Citations:

Pickering B. M., Mitchell S. A., Evans J. R., Willis A. E. (2003) Polypyrimidine tract binding protein and poly r(C) binding protein 1 interact with the BAG-1 IRES and stimulate its activity in vitro and in vivo. Nucleic Acids Res. 31(2):639-646

Pickering B. M., Mitchell S. A., Spriggs K. A., Stoneley M., Willis A. E. (2004) Bag-1 internal ribosome entry segment activity is promoted by structural changes mediated by poly(rC) binding protein 1 and recruitment of polypyrimidine tract binding protein 1. Mol. Cell. Biol. 24(12):5595-5605

Regions with experimentally determined secondary structures:

A region with the experimentally determined secondary structure:

IRESite 2D Struct Id: 10

Version: `1`	Last change: `2007-09-11 00:00:00`
Originaly submitted by: Zuzana Feketová	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):
249-432

The underlying nucleic acid sequence and structure of the mapped region:

AGCGGGCATG ACCGACCCAC CAGGGGCGCC GCCGCCGGCG CTCGCAGGCC GCGGATGAAG AAGAAAACCC GGCGCCGCTC GACCCGGAGC GAGGAGTTGA 
.......... ..((.(((.. ..(((.(((( .((((.(((. .......))) ))))...... ........(( ..((...... (((((..... ..)).))).. 
^249       ^259       ^269       ^279       ^289       ^299       ^309       ^319       ^329       ^339       

CCCGGAGCGA GGAGTTGACC CTGAGTGAGG AAGCGACCTG GAGTGAAGAG GCGACCCAGA GTGAGGAGGC GACCCAGGGC GAAG
..(((..((( ....)))... )))..))... .....((((( (..((..... .))..)))). ))..)).))) ).))).)))) )...
^349       ^359       ^369       ^379       ^389       ^399       ^409       ^419       ^429

Rendering structure of BAG-1 mRNA 184 nt long with energy of -31.80 kcal/mol as calculated by RNAeval using VARNA Java applet with some IRESite improvements (see VARNA modified by IRESite). Hold left mouse button to move structure parts, hold right mouse button to move whole structure, use mouse wheel to zoom. Right mouse-click opens a menu to export into JPG/SVG and many other options.

You need a Java-enabled browser so that modified varsion of VARNA could be started. See http://www.iresite.org/VARNA/ for more details.

Remarks:

The secondary structural model of the Bag-1 IRES was derived by using the DMS, kethoxal and RNase V1
accessibility data to constrain the Mfold algorithm. Chemical treatment was carried out in 0 grades Celsius.
RNase V1 cleavage was performed at room temperature, protocol was adapted form the Ambion protocol.

4.1.1. Enzymes used to characterize at least partially the 2D structure.

Enzyme or a combination of enzymes used in a single experiment with respective buffer:

ss_experiment_with_enzyme_id: 1

The temperature (in degrees of Celsia):
20 The enzymatic method used to determine the 2D structure:
ribonuclease V1

Enzyme or a combination of enzymes used in a single experiment with respective buffer:

Version: 0

pH `7.00`	Li⁺ [mM] `0`	Na⁺ [mM] `0`	K⁺ [mM] `100.00`	Mg²⁺ [mM] `10.00`	Ca²⁺ [mM] `0`
Cl^- [mM] `110.00`	Tris [mM] `10.00`	BSA [mM] `0`	HEPES [mM] `0`	EGTA [mM] `0`	EDTA [mM] `0`
cacodylate [mM] `0`

4.1.2. Chemicals used to characterize at least partially the 2D structure.

Chemical reagent used with its respective buffer:

ss_experiment_with_chemical_id: 1

The temperature (in degrees of Celsia):
0 The chemical reagent used to determine the 2D structure:
DMS

Chemical reagent used with its respective buffer:

Version: 0

pH `7.00`	Li⁺ [mM] `0`	Na⁺ [mM] `0`	K⁺ [mM] `100.00`	Mg²⁺ [mM] `0`	Ca²⁺ [mM] `0`
Cl^- [mM] `100.00`	Tris [mM] `10.00`	BSA [mM] `0`	HEPES [mM] `0`	EGTA [mM] `0`	EDTA [mM] `0`
cacodylate [mM] `0`

Other buffer components and their relative concentrations:

0.83% DMS

Chemical reagent used with its respective buffer:

ss_experiment_with_chemical_id: 2

The temperature (in degrees of Celsia):
0 The chemical reagent used to determine the 2D structure:
kethoxal

Chemical reagent used with its respective buffer:

Version: 0

pH `7.00`	Li⁺ [mM] `0`	Na⁺ [mM] `0`	K⁺ [mM] `100.00`	Mg²⁺ [mM] `0`	Ca²⁺ [mM] `0`
Cl^- [mM] `100.00`	Tris [mM] `10.00`	BSA [mM] `0`	HEPES [mM] `0`	EGTA [mM] `0`	EDTA [mM] `0`
cacodylate [mM] `0`

Other buffer components and their relative concentrations:

0.5% kethoxal

Citations:

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