Contribution of the tyrosines to the structure and function of the human U1A N-terminal RNA binding domain.

RNA binding domains (RBDs) are members of a large family of proteins that share minimal sequence conservation but adopt an alpha beta sandwich global fold. Defining the contributions of specific amino acids to RBD structure and RNA binding is critical to understanding the functions of these proteins. In these experiments with the human U1A N-terminal RNA binding domain (RBD1), the contributions from each of its four tyrosines to protein structure, stability, and RNA binding were measured. Each tyrosine was substituted with phenylalanine and one other selected residue, and the resulting proteins were characterized by chemical denaturation to measure their unfolding free energy, by binding free energies to the wild-type RNA hairpin, and by 19F NMR to probe for structural changes. Features of the protein identified in these experiments include a possible tyrosine/lysine contact in an alpha-helix, which may be an example of an energetically favorable aromatic/amino side chain interaction. One long loop of the protein, which shows unusual 15N backbone and tyrosine side-chain dynamics, is implicated in protein:protein association. The diverse interactions of the four tyrosine residues in the organization of RBD1 illustrate how each member of this family of proteins will have unique molecular details that contribute to function.     

In vitro selection of RNA aptamers that bind special elongation factor SelB, a protein with multiple RNA-binding sites, reveals one major interaction domain at the carboxyl terminus.

The SelB protein of Escherichia coli is a special elongation factor required for the cotranslational incorporation of the uncommon amino acid selenocysteine into proteins such as formiate dehydrogenases. To do this, SelB binds simultaneously to selenocysteyl-tRNA(Sec) and to an RNA hairpin structure in the mRNA of formiate dehydrogenases located directly 3' of the selenocysteine opal (UGA) codon. The protein is also thought to contain binding sites allowing its interaction with ribosomal proteins and/or rRNA. SelB thus includes specific binding sites for a variety of different RNA molecules. We used an in vitro selection approach with a pool completely randomized at 40 nt to isolate new high-affinity SelB-binding RNA motifs. Our main objective was to investigate which of the various RNA-binding domains in SelB would turn out to be prime targets for aptamer interaction. The resulting sequences were compared with those from a previous SELEX experiment using a degenerate pool of the wild-type formiate dehydrogenase H (fdhF) hairpin sequence (Klug SJ et al., 1997, Proc. Natl. Acad. Sci. USA 94:6676-6681). In four selection cycles an enriched pool of tight SelB-binding aptamers was obtained; sequencing revealed that all aptamers were different in their primary sequence and most bore no recognizable consensus to known RNA motifs. Domain mapping for SelB-binding aptamers showed that despite the different RNA-binding sites in the protein, the vast majority of aptamers bound to the ultimate C-terminus of SelB, the domain responsible for mRNA hairpin binding.     

A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.     

Apoptosis Induced by the Nuclear Death Domain Protein p84N5 Is Inhibited by Association with Rb Protein

Rb protein inhibits both cell cycle progression and apoptosis. Interaction of specific cellular proteins, including E2F1, with Rb C-terminal domains mediates cell cycle regulation. In contrast, the nuclear N5 protein associates with an Rb N-terminal domain with unknown function. The N5 protein contains a region of sequence similarity to the death domain of proteins involved in apoptotic signaling. We demonstrate here that forced N5 expression potently induces apoptosis in several tumor cell lines. Mutation of conserved residues within the death domain homology compromise N5-induced apoptosis, suggesting that it is required for normal function. Endogenous N5 protein is specifically altered in apoptotic cells treated with ionizing radiation. Furthermore, dominant interfering death domain mutants compromise cellular responses to ionizing radiation. Finally, physical association with Rb protein inhibits N5-induced apoptosis. We propose that N5 protein plays a role in the regulation of apoptosis and that Rb directly coordinates cell proliferation and apoptosis by binding specific proteins involved in each process through distinct protein binding domains.     

Characterization of the RNA motif responsible for the specific interaction of potato spindle tuber viroid RNA (PSTVd) and the tomato protein Virp1

Viroids are small non-coding parasitic RNAs that are able to infect their host plants systemically. This circular naked RNA makes use of host proteins to accomplish its proliferation. Here we analyze the specific binding of the tomato protein Virp1 to the terminal right domain of potato spindle tuber viroid RNA (PSTVd). We find that two asymmetric internal loops within the PSTVd (+) RNA, each composed of the sequence elements 5′-ACAGG and CUCUUCC-5′, are responsible for the specific RNA–protein interaction. In view of the nucleotide composition we call this structural element an ‘RY motif’. The RY motif located close to the terminal right hairpin loop of the PSTVd secondary structure has an ~5-fold stronger binding affinity than the more centrally located RY motif. Simultaneous sequence alterations in both RY motifs abolished the specific binding to Virp1. Mutations in any of the two RY motifs resulted in non-infectious viroid RNA, with the exception of one case, where reversion to sequence wild type took place. In contrast, the simultaneous exchange of two nucleotides within the terminal right hairpin loop of PSTVd had only moderate influence on the binding to Virp1. This variant was infectious and sequence changes were maintained in the progeny. The relevance of the phylogenetic conservation of the RY motif, and sequence elements therein, amongst various genera of the family Pospiviroidae is discussed.     

The carboxy-terminal domain of the DExDH protein YxiN is sufficient to confer specificity for 23S rRNA

DE x DH proteins are believed to modulate the structures of RNAs and ribonucleoprotein complexes by disrupting RNA helices and RNA-protein interactions. All DE x DH proteins contain a two-domain catalytic core that enables their RNA-dependent ATPase and RNA helicase activities. The catalytic core may be flanked by ancillary domains that are proposed to confer substrate specificity and facilitate the unique functions of individual proteins. The Escherichia coli DE x DH protein DbpA and its Bacillus subtilis ortholog YxiN have similar 75aa carboxy-terminal domains, and both proteins are specifically targeted to 23S rRNA. Here we demonstrate that the carboxy-terminal domain of YxiN is sufficient to confer RNA specificity by characterizing a chimera in which this domain is appended to the core domains of E.coli SrmB, a DE x DH protein with no apparent substrate specificity. Both the RNA-dependent ATPase and RNA helicase activities of the chimera are specifically activated by 23S rRNA and abolished by sequence changes within hairpin 92, a critical recognition element for Y x iN. These data support a model in which the carboxy-terminal domain binds hairpin 92 to target the protein to 23S rRNA.     

Crystal structure of the oligomerization domain of the phosphoprotein of vesicular stomatitis virus

In the replication cycle of nonsegmented negative-strand RNA viruses, the viral RNA-dependent RNA polymerase (L) recognizes a nucleoprotein (N)-enwrapped RNA template during the RNA polymerase reaction. The viral phosphoprotein (P) is a polymerase cofactor essential for this recognition. We report here the 2.3-angstroms-resolution crystal structure of the central domain (residues 107 to 177) of P from vesicular stomatitis virus. The fold of this domain consists of a beta hairpin, an alpha helix, and another beta hairpin. The alpha helix provides the stabilizing force for forming a homodimer, while the two beta hairpins add additional stabilization by forming a four-stranded beta sheet through domain swapping between two molecules. This central dimer positions the N- and C-terminal domains of P to interact with the N and L proteins, allowing the L protein to specifically recognize the nucleocapsid-RNA template and to progress along the template while concomitantly assembling N with nascent RNA. The interdimer interactions observed in the noncrystallographic packing may offer insight into the mechanism of the RNA polymerase processive reaction along the viral nucleocapsid-RNA template.     

A common RNA recognition motif identified within a defined U1 RNA binding domain of the 70K U1 snRNP protein

We have defined the RNA binding domain of the 70K protein component of the U1 small nuclear ribonucleoprotein to a region of 111 amino acids. This domain encompasses an octamer sequence that has been observed in other proteins associated with RNA, but has not previously been shown to bind directly to a specific RNA sequence. Within the U1 RNA binding domain, an 80 amino acid consensus sequence that is conserved in many presumed RNA binding proteins was discerned. This sequence pattern appears to represent an RNA recognition motif (RRM) characteristic of a distinct family of proteins. By site-directed mutagenesis, we determined that the 70K protein consists of 437 amino acids (52 kd), and found that its aberrant electrophoretic migration is due to a carboxy-terminal charged domain structurally similar to two Drosophila proteins (su(wa) and tra) that may regulate alternative pre-messenger RNA splicing.     

Oleosin KD 18 on the surface of oil bodies in maize. Genomic and cDNA sequences and the deduced protein structure

Oleosins are newly discovered, abundant, and small Mr hydrophobic proteins localized on the surface of oil bodies in diverse seeds. So far, most of the studies have been on the general characteristics of the proteins, and only one protein (maize KD 16) has been studied using a cDNA clone containing an incomplete coding sequence. Here, we report the sequences of a genomic clone and a cDNA clone of a new maize oleosin (KD 18). There is no intron in the gene. The 5'-flanking region contains potential regulatory elements including RY repeats, CACA consensus, and CATC boxes, which are presumably involved in the specific expression of the proteins in maturing seeds. The deduced amino acid sequence was analyzed for secondary structures. We suggest that KD 18 of 187-amino acid residues contains three major structural domains: a largely hydrophilic domain at the N terminus, a hydrophobic hairpin alpha-helical domain at the center, and an amphipathic alpha-helix domain at the C terminus. These structural domains are very similar to those of oleosin KD 16. However, the KD 18 and KD 16 amino acid sequences as well as nucleotide sequences are highly similar only at the central domain (72 and 71%, respectively). The similarities are highest at the loop region of the alpha-helical hairpin. These results suggest that KD 18 and KD 16 are isoforms, encoded by genes derived from a common ancestor gene. We propose that the hairpin domain acts as an indispensible internal signal for intracellular trafficking of oleosins during protein synthesis as well as an anchor for oleosins on the oil bodies. The other two domains can undergo relatively massive amino acid substitutions without impairing the structure/function of the oleosins or have evolved to generate oleosins having different functions.     

Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain.

The N-terminal RNA binding domain (RBD) of the human U1A snRNP protein binds tightly and specifically to an RNA hairpin that contains a 10-nucleotide loop. The protein is one of a class of RNA binding proteins that adopts a beta alpha beta beta alpha beta global fold, which in turn forms a four-stranded antiparallel beta-sheet. This sheet forms the primary binding surface for the RNA, as shown by the crosslinking results described here, and in more detail by a recently described co-crystal of this RBD with an RNA hairpin (Oubridge C, et al., 1994, Nature 372:432-438). The RNA hairpin sequence used in the crosslinking experiments, containing 5-iodo-uridine, is a variant of the normal U1 snRNA sequence which is able to form a crosslink with the protein, in contrast to the wild-type sequence, which does not. This single uridine substitution in the 10-nucleotide loop is the site of cross-linking to one tyrosine (Tyr 13) in the beta 1 strand of the U1A N-terminal RBD. This same uridine is also crosslinked to a mutant Tyr 13 Phe RBD, at this Phe 13 substitution.     

Internal targeting signal of the BCS1 protein: a novel mechanism of import into mitochondria.

The BCS1 protein is anchored in the mitochondrial inner membrane via a single transmembrane domain and has an N(out)-C(in) topology. Unlike the majority of nuclear encoded mitochondrial preproteins, the BCS1 protein does not contain an N-terminal targeting sequence. A positively charged segment of amino acids which is located immediately C-terminal to the transmembrane domain acts as an internal targeting signal. In order to function, we postulate that this sequence co-operates with the transmembrane domain to form a tight hairpin loop structure. This loop is translocated across the inner membrane via the MIM/mt-Hsp70 machinery in a membrane potential-dependent manner. This novel mechanism of import and sorting of the BCS1 protein is proposed to represent a more general mechanism used by a number of inner membrane proteins.