Two proteins of the Hsp70 class (Ssb and Ssz1) and one of the J-type class (Zuo1) of molecular chaperones reside on the yeast ribosome, with Ssz1 forming a stable heterodimer with Zuo1. We designed experiments to address the roles of these two distantly related ribosome-associated Hsp70s and their functional relationship to Zuo1. Strains lacking all three proteins have the same phenotype as those lacking only one, suggesting that these chaperones all function in the same pathway. The Hsp70 Ssb, whose peptidebinding domain is essential for its in vivo function, can be crosslinked to nascent chains on ribosomes that are as short as 54 amino acids, suggesting that Ssb interacts with nascent chains that extend only a short distance beyond the tunnel of the ribosome. A ssz1 mutant protein lacking its putative peptide-binding domain allows normal growth. Thus, binding of unfolded protein substrates in a manner similar to that of typical Hsp70s is not critical for Ssz1's in vivo function. The three chaperones are present in cells in approximately equimolar amounts compared with ribosomes. The level of Ssb can be reduced only a few-fold before growth is affected. However, a 50-to 100-fold reduction of Ssz1 and Zuo1 levels does not have a substantial effect on cell growth. On the basis of these results, we propose that Ssbs function as the major Hsp70 chaperone for nascent chains on the ribosome, and that Ssz1 has evolved to perform a nonclassical function, perhaps modulating Zuo1's ability to function as a J-type chaperone partner of Ssb.

ADARs are a family of enzymes, present in all animals, that convert adenosines to inosines within double-stranded RNA. Inosine has different base-pairing properties from adenosine, and thus, editing alters RNA structure, coding potential and splicing patterns. The first identified ADAR substrates were edited in codons, and ADARs were presumed to function primarily in proteome diversification. Although this is an important ADAR function, especially in the nervous system, editing in coding sequences is rare compared to editing in non-coding sequences. Introns and untranslated regions of mRNA are the primary non-coding targets, but editing also occurs in small RNAs, such as miRNAs. Although the role of editing in non-coding sequences remains unclear, ongoing research suggests functions in the regulation of a variety of post-transcriptional processes. Basics of RNA editing by ADARsRNA editing is the alteration of RNA by nucleotide modification, insertion or deletion (reviewed in [1]). In metazoans, a common type of RNA editing is a nucleotide modification involving the hydrolytic deamination of adenosine (A) to inosine (I). This type of editing is catalyzed by the Adenosine Deaminases that act on RNA (ADARs) family [2-4], which target RNA that is completely, or largely, double-stranded. ADARs are likely present in all animals and have been studied in squids, worms, flies and mammals. A single organism encodes between 1-3 ADARs, and all family members contain a highly conserved catalytic domain at their C-terminus, and variable numbers of N-terminal dsRNA binding motifs (dsRBMs). Whereas ADARs are essential in mammals, invertebrates that lack ADARs are viable, but exhibit behavioral defects [5][6][7][8].Inosine is recognized as guanosine (G) by the translation and splicing machineries, and thus, ADARs can alter the protein-coding information of an mRNA. In addition, because inosine prefers to pair with cytidine (C), ADARs destabilize dsRNA by changing AU base-pairs to IU mismatches [9,10], or increase its stability by changing AC mismatches to IC base-pairs [11]; while other changes to RNA structure have not been documented, conceivably, ADARs could promote more complex structural rearrangements as well. In choosing which adenosines to target, ADARs show slight sequence preferences [12,13], and in addition, the number of adenosines targeted in a dsRNA is dependent on its length and structure. For example, in vitro studies show that duplexes of about 15-40 bp are edited selectively, at very few sites,

Edited by Charles E. SamuelRNA editing is a cellular process that precisely alters nucleotide sequences, thus regulating gene expression and generating protein diversity. Over 60% of human transcripts undergo adenosine to inosine RNA editing, and editing is required for normal development and proper neuronal function of animals. Editing of one adenosine in the transcript encoding the glutamate receptor subunit B, glutamate receptor ionotropic AMPA 2 (GRIA2), modifies a codon, replacing the genomically encoded glutamine (Q) with arginine (R); thus this editing site is referred to as the Q/R site. Editing at the Q/R site of GRIA2 is essential, and reduced editing of GRIA2 transcripts has been observed in patients suffering from glioblastoma. In glioblastoma, incorporation of unedited GRIA2 subunits leads to a calcium-permeable glutamate receptor, which can promote cell migration and tumor invasion. In this study, we identify adenosine deaminase that acts on RNA 3 (ADAR3) as an important regulator of Q/R site editing, investigate its mode of action, and detect elevated ADAR3 expression in glioblastoma tumors compared with adjacent brain tissue. Overexpression of ADAR3 in astrocyte and astrocytoma cell lines inhibits RNA editing at the Q/R site of GRIA2. Furthermore, the double-stranded RNA binding domains of ADAR3 are required for repression of RNA editing. As the Q/R site of GRIA2 is specifically edited by ADAR2, we suggest that ADAR3 directly competes with ADAR2 for binding to GRIA2 transcript, inhibiting RNA editing, as evidenced by the direct binding of ADAR3 to the GRIA2 pre-mRNA. Finally, we provide evidence that both ADAR2 and ADAR3 expression contributes to the relative level of GRIA2 editing in tumors from patients suffering from glioblastoma.Adenosine deaminases that act on RNA (ADARs) 2 catalyze the hydrolytic deamination of adenosine residues within double-stranded RNA (dsRNA) structures that form by base pairing of nearby complementary sequences (1-3). This RNA editing event creates a non-canonical nucleoside, inosine, which base pairs similarly to guanosine (4). RNA editing affects gene expression through modification of codons to create novel protein isoforms (5). Furthermore, editing can alter microRNA and siRNA binding sites, splice acceptor or splice donor sites, or RNA structure and stability (6 -9). A majority of the targets of RNA editing are found in the nervous system, and RNA editing is critical for maintaining proper neuronal function (10). Furthermore, transcripts encoding proteins involved in neurotransmission are often targets of RNA editing, which alters the protein amino acid sequence and physiological function of these ion channels and receptors (11,12).In mammals, three ADAR proteins, ADAR1, ADAR2, and ADAR3, and two ADAR-like proteins, ADAD1 and ADAD2, have been identified (13-17). The ADAR and ADAR-like protein family members contain several common modular domains that are important for function (18,19). Most strikingly, the human ADAR and ADAR-like proteins contain at least one ds...

SUMMARY Inadequate adenosine-to-inosine editing of noncoding regions occurs in disease, often uncorrelated with ADAR levels, underscoring the need to study deaminase-independent control of editing. C. elegans have two ADAR proteins, ADR-2 and the theoretically catalytically inactive ADR-1. Using high-throughput RNA sequencing of wild-type and adr mutant worms, we expanded the repertoire of C. elegans edited transcripts over 5-fold and confirmed that ADR-2 is the only active deaminase in vivo. Despite lacking deaminase function, ADR-1 affects editing of over 60 adenosines within the 3′ UTRs of 16 different mRNAs. Furthermore, ADR-1 interacts directly with ADR-2 substrates, even in the absence of ADR-2; and mutations within its dsRNA binding domains abolished both binding and editing regulation. We conclude that ADR-1 acts as a major regulator of editing by binding ADR-2 substrates in vivo and raises the possibility that other dsRNA binding proteins, including the inactive human ADARs, regulate RNA editing by deaminase-independent mechanisms.