Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo-and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent. Double-stranded RNAs (dsRNAs)2 in eukaryotes are subject to a variety of processing reactions, including cleavage by the RNase III family members Drosha and Dicer in the micro RNA and small interfering RNA gene-silencing pathways and editing by members of the ADAR (adenosone deaminase that acts on RNA) enzyme family (1, 2). This latter reaction involves the hydrolytic deamination of adenosine (A) to inosine (I) within the context of dsRNA. Editing events of this type have been demonstrated in both cellular and viral transcripts and have been shown to function in altering the coding properties of the edited RNAs. For example, the life cycle of the Hepatitis ␦ virus is regulated by an editing event in the anti-genome in which a UAG stop codon is converted to a UIG tryptophan codon (3). An A to I modification is involved in the functional regulation of a growing number of cellular factors. These include the tissue-specific editing of the serotonin 5-HT2C receptor, which results in a reduction in response to serotonin agonists (4). Transcripts for subunits of the neural-specific AMPA class of glutamate-gated (GluR) ion channels undergo A to I modification at two positions, the Q/R and R/G editing sites, that affect the properties of the resulting channels (5, 6). In addition to the editing of these and other neuronal transcripts to effect codon changes, one deaminase family member, ADAR2, has been shown to autoregulate its own expression by the creation of a 3Ј-splice site (CAA to CAI) (7). Despite the identification of these editing substrates, the global role of A to I modification in higher eukaryotes remains unclear. Measurement of inosine levels in RNA isolated from rat tissue suggests a greater level of editing than indicated by known RNA substrates (8). A cloning protocol that depended upon an inosine-specific cleavage of RNA detected a large number of editing sites in non-coding regions of RNAs from Caenorhabditis elegans and humans that included sites in 5Ј-and 3Ј-untranslated regions and introns (9). Recent bioinformatic studies have suggested the presence of more than 12,000 editing sites corresponding to non-coding regions of t...
We uncovered a new pathway of interplay between calreticulin and myocyte-enhancer factor (MEF) 2C, a cardiac-specific transcription factor. We establish that calreticulin works upstream of calcineurin and MEF2C in a Ca2+-dependent signal transduction cascade that links the endoplasmic reticulum and the nucleus during cardiac development. In the absence of calreticulin, translocation of MEF2C to the nucleus is compromised. This defect is reversed by calreticulin itself or by a constitutively active form of calcineurin. Furthermore, we show that expression of the calreticulin gene itself is regulated by MEF2C in vitro and in vivo and that, in turn, increased expression of calreticulin affects MEF2C transcriptional activity. The present findings provide a clear molecular explanation for the embryonic lethality observed in calreticulin-deficient mice and emphasize the importance of calreticulin in the early stages of cardiac development. Our study illustrates the existence of a positive feedback mechanism that ensures an adequate supply of releasable Ca2+ is maintained within the cell for activation of calcineurin and, subsequently, for proper functioning of MEF2C.
Recognition of the 3 splice site in mammalian introns is accomplished by association of the splicing factor U2AF with the precursor mRNA (pre-mRNA) in a multiprotein splicing commitment complex. It is well established that this interaction involves binding of the large U2AF65 subunit to sequences upstream of the 3 splice site, but the orientation of the four domains of this protein with respect to the RNA and hence their role in structuring the commitment complex remain unclear and the basis of contradictory models. We have examined the interaction of U2AF65 with an RNA representing the 3 splice site using a series of U2AF deletion mutants modified at the N terminus with the directed hydroxyl radical probe iron-EDTA. These studies, combined with an analysis of extant high resolution x-ray structures of protein⅐RNA complexes, suggest a model whereby U2AF65 bends the pre-mRNA to juxtapose reactive functionalities of the pre-mRNA substrate and organize these structures for subsequent spliceosome assembly.Removal of non-coding intron sequences from pre-mRNAs 1 in eukaryotes involves two sequential transesterifications. In the first step, the branch point adenosine within the intron carries out a nucleophilic displacement at the 5Ј splice site, producing the 5Ј exon and lariat intermediate. The liberated 5Ј exon attacks the 3Ј splice site to yield ligated exon and lariat intron products. Both reactions are catalyzed by the spliceosome: a large (ϳ60 S) ribonucleoprotein assembly. The spliceosome consists of the U1, U2, and U4/U5/U6 snRNPs (small nuclear ribonucleoprotein particles), each containing a unique snRNA and associated proteins as well as non-snRNP splicing factors (1-3). Assembly of the spliceosome on the pre-mRNA proceeds through the formation of several complexes and is directed by conserved sequences at the 5Ј and 3Ј splice sites as well as other sequences in the pre-mRNA; recognition of the 3Ј splice site is closely coupled to recognition of the proximal branch region and polypyrimidine tract within the intron. Regulation of this assembly process results in differential splice site usage, and the resulting patterns of alternative splicing are a major source of proteome diversity in higher eukaryotes (4, 5).Commitment of a pre-mRNA to the splicing pathway involves the ATP-independent formation of the early or commitment complex on the pre-mRNA substrate (1-3). In mammals, this complex includes U1 snRNP, tightly associated with the 5Ј splice site, as well as non-snRNP protein factors. These proteins include the heterodimer U2AF, containing large (U2AF65) and small (U2AF35) subunits, which binds to the polypyrimidine tract and 3Ј splice site, the branch-binding protein SF1, and members of the SR protein family (6 -11). Following the formation of the commitment complex, U2 snRNP is recruited to the pre-mRNA in an ATP-dependent process. This association involves the formation of a duplex between U2 snRNA and the pre-mRNA branch region, which bulges out the branch adenosine, specifying it as the nucleophile fo...
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