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RNA editing is an important mechanism for regulating genetic plasticity through the generation of alternative protein products from a single structural gene. Substitutional RNA editing employs a variety of genetic mechanisms, the biochemical basis of which has been elucidated following the development of in vitro assays that recapitulate important elements of this process. There are two types of substitutional RNA exist in mammals, namely A-to-I and C-to-U RNA editing. The best-characterized example of C-to-U RNA editing involves the nuclear transcript encoding intestinal apolipoprotein B (apo B). Apo B RNA editing changes a CAA to a UAA stop codon, generating a truncated protein, apoB48. The functional complex includes a minimal core composed of apobec-1 and ACF, that function as an adaptor protein by binding both the deaminase and the RNA substrate. The RNA binding proteins also include CUGBP2 which along with Apobec-1 binds to the consensus binding sequence UUUN (A/U) U, present in c-myc, VEGF and Cyclooxygenase-2 (COX2).

Myotonic dystrophy (DM), a disease associated with an unstable trinucleotide CTG repeat located in the myotonin protein kinase gene (DMPK), whereby expanded CTG (CUG) repeats result in gain of function of specific RNA-binding proteins that could regulate RNA processing in multiple tissues. A novel RNA-binding protein, CUGBP1, which specifically binds to CUG repeat sequences. CUGBP has been suggested as a candidate for an RNA-binding protein that regulates a number of RNAs by binding to CUG repeats in regulatory regions of different mRNAs. (CUGBP) exhibited no binding to an RNA oligonucleotide of triplet repeats of the same length but having a different sequence, CGG. The CUG-binding protein was localized to the cytoplasm, whereas double-stranded DNA binding proteins were localized to the nuclear extract.

Rat CUG-BP1 (513 aa, mouse/human 486 aa, chromosome 11p11) also known as CUG-BP1, RNA-binding protein BRUNOL-2, Deadenylation factor CUG-BP, 50 kDa Nuclear polyadenylated RNA-binding protein, EDEN-BP, is expressed in most tissues.

A second member of the family of CUG-binding proteins, CUGBP2 or elav-type ribonuclear protein ETR3, which is highly expressed in heart and is able to interact with CUG repeats. Mouse Etr3 was found to have a molecular weight of 50 kD and displayed a high level of homology with CUGBP1 protein. Organization of the RNA-binding domains within the Etr3 molecule was similar to one within CUGBP. ETR3 is highly expressed in the heart but is undetectable in other tissues. CUGBP2/ NAPOR/ Brunol 3/ ETR-3, a novel RNA-binding protein (54kD) found as a component of holoenzyme Apolipoprotein B (apo B) beside apobec-1 and ACF. It plays a role in apo B mRNA editing by forming a regulatory complex with the three components of the minimal editing enzyme, apobec-1, ACF and apo B RNA. Posttranscriptional C to U RNA editing of apo B creates an in-frame stop codon in the edited transcript that in turn results in translation of a truncated protein apo B48. Apo B mRNA editing takes place in mammalian small intestine, it is accordingly an important physiological role in mammalian lipoprotein metabolism. The CUGBP2/ NAPOR is a 509aa long protein (Chr 10p13-p14) exists in three isoforms NAPOR 1, 2 and 3. NAPOR 1 and 2 are same except additional 19aa residues upstream of NAPOR 1, whereas NAPOR 3 differs from other two because of its 5 extra amino residues upstream of N-terminus of NAPOR 2, also 6aa between its second and third RNA binding domains are absent from NAPOR 3. NAPOR 3 is neuron specific in expression whereas the other two are ubiquitous.

Apobec-1, 236aa protein in human (chr 12p13.1) an RNA specific cytidine deaminase, is essential but not efficient for apo B editing activity, there being a requirement for other protein factor. Apobec-1 is a dimer with the composite active site assembled through interaction of each monomer; In addition it is an RNA-binding protein that binds to the consensus sequence UUUN (A/U) U located within the terminal loop of apo B RNA. But finally it forms the minimal component of the core-editing enzyme along with ACF.

ACF (apobec-1 complementation factor) exists in three isoforms; the isoform 3 is a 594aa protein (65kD), when added with apobec-1 it reconstitute the editing of an apo B mRNA template. It is found to co-localize with apobec-1 and CUGBP2. It was demonstrated that CUGBP2 co-fractionates with ACF in bovine liver S-100 extracts and that its distribution in the most enriched fractions closely matches with ACF, thus leading to the assembly to apo B holoenzyme.

 

Items	Antigen peptide location	Antibody Host	*Expected Ab Crossreactivity	Aff. Pure IgG/Mab Cat # (100 ug)	* Control Peptide Cat# (100 ug)
CUGBP1	R, CUGBP1	m, momo	R, M, H, B		CUGBP11-M
CUGBP2	h, 14 aa ~CT (cytoplasmic)	Rb	h, m, r	CUGBP21-A	CUGBP21-P
APOBEC1	h, 15 aa ~NT (extracellular)	Rb	h, m?	APOBEC11-A	APOBEC11-P
ACF	h, 14 aa ~Internal	Rb	h, m, r	ACF11-A	ACF11-P
Control Rabbit IgG		For use in ELISA, Western, Immunohisto.		20009-1 (1 mg)

Rb=rabbit; m=mouse; r=rat; h=human; s=sheep; b=bovine; c=chicken; d=dog; ~CT or ~NT=near C or N-terminus. EC=Extracellular; CL=Cytoplasmic loop.

** Expected antibody crossreactivity information is mostly based upon high (>70%) sequence conservation of antigenic/control peptides in various species. When antibody crossreactivity has actually been experimentally confirmed in various species, it will be mentioned in the appropriate data sheets.

"Neat Antisera or antisera" are the unpurified antiserum and it is suitable for ELISA and Western.
"Affinity pure" IgG may be more suitable for immunohistochemical (IHC) applications and to reduce background in most immunological applications including ELISA and Western.
"Control peptides" can not be used for Western as they are very short peptides. They are intended for ELISA or antibody blocking studies to establish antibody specificity.
Western blot +ve protein controls, where available, are semi-pure, pure or recombinant proteins that are formulated in SDS-PAGE sample buffer. They are recommended to be used for Western (load 10 ul/lane) for visulaization with antibodies.

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vb@vincibiochem.it

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