IRESite_Id:8 summary record describing characteristics of Rbm3 IRES

IRESite: The database of experimentally verified IRES structures

The nucleic acid data:

IRESite Id: `8`	Version: `11`
Originaly submitted by: Zuzana Feketová	Submission date: `2005-06-27 00:00:00`
Reviewed by: Martin Pospíšek	Last change: `2009-04-08 16:34:57`

IRESite record type:

natural_transcript

The shape of the nucleic acid molecule translated: `linear`	The quality of the mRNA/+RNA sequence: `possibly_wrong`
The abbreviated name of the virus/gene coding for this mRNA/+RNA molecule: `Rbm3`	The genetic origin of this natural mRNA/+RNA: `nuclear`

The GenBankId GI:# number of exactly this mRNA/+RNA sequence:
15983756

The mRNA/+RNA description:

Mus musculus RNA-binding motif protein 3 mRNA, complete cds. Actually reported to be a cloning artifact, a
merge between Thoc1 mRNA and Rbm3 mRNA.

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

CACGAGGCAT TACCCAGTAG ACTCTTACAC TGAACCTCCC AATCCTAACT TAGTACATGC ATACAATAAA TGCTCACTAA GTACTGATAT ATGATTAAAA 
^1         ^11        ^21        ^31        ^41        ^51        ^61        ^71        ^81        ^91        

CACTTGAAAA CAAACAAACT GTAATGTACA TGACATAATC GTGATGAGCA GTAAGTAGGA ACATCTTCTT GGCAAAGGAA GGAGTCAAAC AATGAGCAGG 
^101       ^111       ^121       ^131       ^141       ^151       ^161       ^171       ^181       ^191       

GGCTGTGAGT GTGGGTCAGT GGTAGCACAG CATGCATGAG GCCCTATATT TGGTCTCTAG CAACACACAT AAAAGATGAA CAAAACCAGA CAGAGGAAGA 
^201       ^211       ^221       ^231       ^241       ^251       ^261       ^271       ^281       ^291       

GGTATGTCGA GAGTCTTAAA TAAAACAGGT CAGATCTAAA ACAACTTTTA GGAGAAACAG AAAGATACTT ACTTTCGGTC ACTCCCCCAA TAGCAAGAGA 
^301       ^311       ^321       ^331       ^341       ^351       ^361       ^371       ^381       ^391       

AATAATAGCT AAAACGTTCT CACATGCGGA ATGATTTATA ATTTCTTCTT CCAGAACACC TCTGAAAGCT TGGTCAAGGG TACATTTTTT TTCATTTTCA 
^401       ^411       ^421       ^431       ^441       ^451       ^461       ^471       ^481       ^491       

CTGCCAGGTA ACTGACTGAA GGCAGTCAAC AACGGCTTGA TATTTTTGTT ATTCAAGGCT TCTCTGGTAG ATTTCGTAAA TCGCGTCCGC GCCTCCGGCA 
^501       ^511       ^521       ^531       ^541       ^551       ^561       ^571       ^581       ^591       

AACTGAAAAG CGCTTTATCA GGCGTCTTCC CGCGCCGCAG TCTCTCTGTT CTCCCGGTTC CTTCGAGCTC GTCGTCTCTG CCGTCCTCTG ACTTTTAATT 
^601       ^611       ^621       ^631       ^641       ^651       ^661       ^671       ^681       ^691       

TCCAGGACTT GCCTTCTGCC ATGTCGTCTG AAGAAGGGAA ACTCTTCGTA GGAGGGCTCA ACTTCAACAC CGATGAACAG GCACTTGAAG ACCACTTCAG 
^701       ^711       ^721       ^731       ^741       ^751       ^761       ^771       ^781       ^791       

CAGCTTTGGG CCTATCTCTG AGGTGGTTGT TGTCAAGGAC CGGGAGACTC AAAGATCCCG GGGTTTTGGC TTCATCACCT TCACAAACCC AGAGCATGCC 
^801       ^811       ^821       ^831       ^841       ^851       ^861       ^871       ^881       ^891       

TCAGATGCGA TGAGAGCTAT GAATGGAGAG TCCCTGGATG GGCGCCAAAT CCGAGTTGAT CATGCAGGAA AGTCTGCCAG GGGATCCAGA GGGGGTGCCT 
^901       ^911       ^921       ^931       ^941       ^951       ^961       ^971       ^981       ^991       

TTGGTGGGCG TGGTCGCAGT TACTCTAGAG GTGGTGGAGA CCAGGGATAT GGAAGTGGAA GATATGACAG TCGTCCTGGA GGATATGGAT ATGGGTATGG 
^1001      ^1011      ^1021      ^1031      ^1041      ^1051      ^1061      ^1071      ^1081      ^1091      

GCGGTCTAGA GACTACAGTG GCAGCCAGGG TGGCTATGAC CGCTACTCAG GAGGAAATTA CAGAGACAAT TATGACAACT GAGATGGGGC ATGCACACAA 
^1101      ^1111      ^1121      ^1131      ^1141      ^1151      ^1161      ^1171      ^1181      ^1191      

AATATACACA AGGAATAACA CTTCTGATCC AGGATCGTCC TTGCAAATTG CTGTATTTAT AAAGATTTTT GGAGCTGCGC TCAAACGTCT GTTTTAGTAC 
^1201      ^1211      ^1221      ^1231      ^1241      ^1251      ^1261      ^1271      ^1281      ^1291      

ATCAAGTTCT ATTTTTGAAT TGAGCTCCCA AGGTAGTTTG TTAAAGAACT TTTAGAAAGC TCCATGTGTT CTTTAAACAT TTTTTCCCTT TTAAAAACAA 
^1301      ^1311      ^1321      ^1331      ^1341      ^1351      ^1361      ^1371      ^1381      ^1391      

ATTTTAAGAC ATTTGTTACA GCCCCTCGTG
^1401      ^1411      ^1421

Credibility of mRNA sequence:

end-to-end_sequence_reverse_engineered_and_should_match_experiment

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:
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:
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 Neuro-2a [N2a] (ATCC CCL-131)

Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:

ORF

ORF position:

1

Version: `2`	Last change: `2009-03-16 16:14:09`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Pospíšek

The abbreviated name of this ORF/gene:
Rbm3

The description of the protein encoded in this ORF:
Mus musculus RNA-binding motif protein 3 mRNA

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):

721-1182

Remarks:

The whole 5'UTR was not tested for promoter activity. The only parts tested were:
- the 100nt long region containing the 22-nt IRES module (fragment III)
- the 22-nt module itself (-286 to -265, i.e. bases 435-456 in IRESite annotation)
and none of these contained promoter activity.

The Rbm3 5'leader sequence contains 13 uORF and none of them overlapped the Rbm3 coding sequence. Seven of the
uAUGs contain a purine at position -3 and might be therefore in good context to function as initiation codons.

Sequence comparisons of the Rbm3 5'leader identified several matches with different ESTs, which may indicate
that there are several alternatively spliced variations of the Rbm3 5'leader. Reporter mRNAs containing the
Rbm3 5'leader are translated relatively efficiently, the full length 5'leader has more IRES activity than
5'deleted fragments that contain fewer uORFs. One uORF overlaps the 22-nt IRES module and its translation
seems to mask an inhibitory activity.

Possible presence of a cryptic promoter was ruled out using promoter-less plasmid (Chappel et al., 2003 Fig.
2A).

Baranick et al. (2008) in Supplementary Figure 11 report that the sequence reported as Rbm3 IRES is a cloning
artifact and is actually a reverse-complementary sequence of Thoc1 mRNA. Moreover, they report aberrantly
spliced transcripts.

Citations:

Chappell S. A., Owens G. C., Mauro V. P. (2001) A 5' Leader of Rbm3, a Cold Stress-induced mRNA, Mediates Internal Initiation of Translation with Increased Efficiency under Conditions of Mild Hypothermia. J. Biol. Chem. 276(40):36917-36922

Chappell S. A., Mauro V. P. (2003) The internal ribosome entry site (IRES) contained within the RNA-binding motif protein 3 (Rbm3) mRNA is composed of functionally distinct elements. J. Biol. Chem. 278(36):33793-33800

Baranick B. T., Lemp N. A., Nagashima J., Hiraoka K., Kasahara N., Logg C. R. (2008) Splicing mediates the activity of four putative cellular internal ribosome entry sites. Proc. Natl. Acad. Sci. U. S. A. 105(12):4733-4738

The rRNA complementarities:

The rRNA complementarity:

Version: `6`	Last change: `2006-01-15 00:00:00`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Pospíšek

The organism where this rRNA:RNA interaction occurred:

Mus musculus

GenBankId of the rRNA that interacts with this mRNA/+RNA:
53990

The name of the rRNA interacting with this mRNA/+RNA:
18S

The base range located on rRNA where this interaction occurs in 3'-5' direction.
819-808

The number of bases within that range which actually do interact.
9

The experimental validation of this interaction between mRNA and rRNA in these regions is available:
no

The base range located on mRNA where this interaction occurs in 5'-3' direction.
439-448

Remarks:

                  456                 435
                  >                     <
IRES:          3' AAGACC-TTCTTCTTTAATATTT 5'
                           ::: ::::::
 18S:          5' TT-TACTTTGAAAAAATTAGAGT 3'
                           ^        ^
                          808      819

The above schema represents the 22-nt IRES module and predicted interaction of nucleotides 439-448 of Rbm3
mRNA with bases 819-808 of 18S rRNA (GI:53990). 9 base-pairs are denoted by colons.

The 22nt IRES module ranging from 435-456 was tested to bind purified 40S ribosomal subunit and ribosomes, but
it was not tested yet whether the complementary sequence match contributes to the binding of ribosomes.
Several other complementary sequence matches between Rbm3 5'UTR and mouse 18S rRNA were also predicted (none
of these sequences overlap with the 22nt IRES module):

rRNA in 3'-5'direction           mRNA in 5'-3'direction
1734-1718                        28-44
62-50                            49-62
224-217                          66-73
900-889                          107-118
1020-1001                        159-178
1249-1242                        182-189
1538-1526                        215-228
1368-1360                        284-292
1830-1821                        341-350
1019-1010                        411-420
1632-1619                        423-436
88-78                            493-504
151-142                          548-557
90-75                            636-651
1828-1816                        690-702

All sequence matches listed above were predicted in-silico and none of them was experimentally tested.

IRESs:

IRES:

Version: `11`	Last change: `2009-04-08 16:34:57`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Pospíšek
The IRES name: `Rbm3`

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

435-456

Conclusion:
disproved_IRES

How IRES boundaries were determined:
experimentally_determined

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

TTTATAATTT CTTCTTCCAG AA
^435       ^445       ^455

Remarks:

IRES activity was studied in transiently transfected cells and also T7 bicistronic (capped) transcripts in C6
cell-free lysates. Integrity of the mRNAs was verified by Northern blot.
435-456 is a 22-nt IRES module, which functions as IRES when it is tested in isolation. This 22-nt IRES module
is part of a larger but not further described IRES, containing at least 9 cis-acting sequences. The whole IRES
appears within the 5'UTR of Rbm3 mRNA.

Experimentally tested were: the 22-nt IRES module, 5' and 3' IRES inhibitory sequences and IRES enhancer, all
spanning the region 424-493 nt on mRNA:

                  22-nt IRES module
               >435______________456<
               |                    |
    AUGCGGAAUGAUUUAUAAUUUCUUCUUCCAGAA.......GAAAGCUUGG.......GUACAUUUUUUUUC
    ^______uORF______^                      ^        ^       ^            ^
    424            441                      464    473       480        493
       5'inhibitor                           3'enhancer       3'inhibitor


Baranick et al., 2008 and they postulate that most of the "IRES" activity observed so far was due to the
splice artifacts (Fig. 3D).

Citations:

RNA:protein interactions:

The RNA:protein interaction:

Version: `4`
Originaly submitted by: Zuzana Feketová	Reviewed by: Martin Pospíšek

The description of the protein interacting with the RNA:
not specified protein

The function of the protein interaction:

not known

The organism where this RNA:protein interaction occurs:

Mus musculus Neuro-2a [N2a] (ATCC CCL-131)

Remarks:

Interacting protein was not further described. RNP complexes were detected using mobility shift assay. The
22-nt IRES module, used as the probe was incubated with extracts from NIH-3T3, C6 and N2a cell lines. In the
N2a lysate two RNP complexes formed. Interactions were shown to be sequence specific.

Citations:

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