The interferon-induced protein kinase DAI, the double-stranded RNA (dsRNA)-activated inhibitor of translation, plays a key role in regulating protein synthesis in higher cells. Once activated, in a process that involves autophosphorylation, it phosphorylates the initiation factor eIF-2, leading to inhibition of polypeptide chain initiation. The In mammalian cells, a regulatory mechanism involving an RNA-activated protein kinase and the eukaryotic initiation factor 2 (eIF-2) has been intensively studied. This initiation factor forms a ternary complex with GTP and Met-tRNAF and delivers the initiator tRNA to the ribosomal site of protein synthesis initiation. Discharged eIF-2 is subsequently released as a complex with GDP which must be replaced with GTP to permit the formation of another ternary complex in preparation for a further round of initiation. The factor is composed of three dissimilar subunits, cx, I, and -y. Phosphorylation of a single residue, serine-51 of the a subunit, inhibits translation by trapping a second initiation factor, the guanosine nucleotide exchange factor (or eIF-2B), which is required to catalyze the substitution of GTP for GDP in the discharged eIF-2 complex. Phosphorylation of sufficient eIF-2 can sequester all of the guanosine nucleotide exchange factor, thereby preventing eIF-2 recycling and halting the initiation pathway.In mammals, two protein kinases are capable of phosphorylating the a subunit of eIF-2 in this way (reviewed in references 20, 37, and 46). One of them, the heme-controlled repressor, is found chiefly in reticulocytes. It is activated by the absence of hemin, as well as by other stimuli, and serves to prevent the accumulation of globin in the absence of iron or heme. A second kinase, the double-stranded RNA-activated inhibitor (DAI; also referred to as P1 kinase, p68 kinase, P1/eIF-2a kinase, and PKdS, etc.) is present in a wide range of tissues. DAI is an important element in the host antiviral response, and its synthesis is induced at the transcriptional level by interferon (reviewed in references 21, 54, 56, and 59). The enzyme is ribosome associated (11,34) (8,24,26,36,47,60). It has also been implicated in cellular differentiation (23,52), in the inhibition of cell proliferation (6, 51), in the heat shock response (10), and possibly in transcriptional induction (61, 64). Moreover, in yeast cells, the related protein kinase GCN2 mediates the growth response to amino acid starvation (9). As its name implies, DAI is activated by doublestranded RNA (dsRNA). Other polyanions such as heparin can also activate it, while small, highly structured RNA molecules such as adenovirus VA RNA suppress its activation (38). Thus, DAI is a pivotal cellular regulatory enzyme whose level and activity are modulated by factors of both viral and cellular origin.The interactions between DAI and its RNA effectors are complicated and incompletely understood. The kinase is activated by dsRNA but not by DNA or DNA-RNA hybrids (22,32,35,58). Single-stranded RNA, either synthetic or natur...

Human hnRNP A1 is a versatile single-stranded nucleic acid-binding protein that functions in various aspects of mRNA maturation and in telomere length regulation. The crystal structure of UP1, the amino-terminal domain of human hnRNP A1 containing two RNA-recognition motifs (RRMs), bound to a 12-nucleotide single-stranded telomeric DNA has been determined at 2.1 Å resolution. The structure of the complex reveals the basis for sequence-specific recognition of the single-stranded overhangs of human telomeres by hnRNP A1. It also provides insights into the basis for high-affinity binding of hnRNP A1 to certain RNA sequences, and for nucleic acid binding and functional synergy between the RRMs. In the crystal structure, a UP1 dimer binds to two strands of DNA, and each strand contacts RRM1 of one monomer and RRM2 of the other. The two DNA strands are antiparallel, and regions of the protein flanking each RRM make important contacts with DNA. The extensive protein-protein interface seen in the crystal structure of the protein-DNA complex and the evolutionary conservation of the interface residues suggest the importance of specific protein-protein interactions for the sequence-specific recognition of single-stranded nucleic acids. Models for regular packaging of telomere 3 overhangs and for juxtaposition of alternative 5 splice sites are proposed.[Key Words: hnRNP A1; telomere; RNA-recognition motif; x-ray crystallography]Received February 11, 1999; revised version accepted March 9, 1999.Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is one of the most abundant and best-studied components of hnRNP complexes. Together with other hnRNP proteins, hnRNP A1 packages nascent pre-messenger RNA (pre-mRNA) for processing in the nucleus (for review, see Dreyfuss et al. 1993;McAfee et al. 1997). Although it accumulates predominantly in the nucleus, hnRNP A1 shuttles continuously between the nucleus and the cytoplasm (Piñ ol-Roma and Dreyfuss 1992). Nuclear localization and import-export signals within hnRNP A1 have been mapped to a 38 amino acid sequence, known as M9, located near the carboxyl terminus of the protein (for review, see Izaurralde and Adam 1998). Because hnRNP A1 associates with poly(A) + RNA both in the nucleus and in the cytoplasm, and injection of hnRNP A1 with an intact M9 region into frog oocytes inhibits mRNA export, it is likely that hnRNP A1 is involved in transporting mature mRNA from the nucleus to the cytoplasm.Nuclear hnRNP A1 and other closely related hnRNP A/B proteins can modulate the use of alternative 5Ј splice sites in a concentration-dependent manner. hnRNP A1 activates distal 5Ј splice sites and promotes alternative exon skipping both in vitro and in vivo (Fu et al. 1992;Mayeda and Krainer 1992;Mayeda et al. 1993;Cá ceres et al. 1994;Yang et al. 1994). The alternative splicing activity of hnRNP A1 counteracts that of members of the serine-arginine (SR) family of splicing factors, such as SF2/ASF and SC35. The relative levels or activities of these two classes of antagonistic factors can dete...

Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and-in certain situations-nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.Pre-mRNA splicing takes place within the spliceosome, a macromolecular complex composed of four small nuclear ribonucleoprotein particles (snRNPs) (U1, U2, U4/U6, and U5) and approximately 50 to 100 polypeptides (32). The SR proteins are an extensively characterized family of structurally and functionally related non-snRNP splicing factors that are essential for constitutive splicing and that also influence alternative splicing regulation in higher eukaryotes (5,19,58).The SR proteins are involved in multiple steps of the constitutive splicing reaction. They promote the recruitment of the U1 snRNP to the 5Ј splice site (14,26,29) and of the U2AF splicing factor and U2 snRNP to the branch site or 3Ј splice site (57, 62) and also facilitate the recruitment of the U4/U6-U5 tri-snRNP at later stages of the splicing reaction (51). The SR proteins also bridge pairs of 5Ј and 3Ј splice sites via RS domain-mediated protein-protein interactions, which can occur across an exon or an intron (exon definition or intron definition) (50, 62). The SR proteins are also important regulators of alternative splicing, and their activity is subject to antagonism by members of the hnRNP A/B family of proteins. Increased levels of SR proteins usually promote the selection of proximal alternative 5Ј splice sites both in vitro and in vivo, whereas an excess of hnRNPs A/B proteins usually results in the selection of distal 5Ј splice sites (8,38,63). Tissue-specific variations in the total and relative amounts of SR proteins and in the molar ratio of SF2/ASF to its antagonist, hnRNP A1, support the notion that the relative levels and activities of these two families of antagonistic factors may be crucial in regulating th...

Telomerase is a ribonucleoprotein enzyme complex that reverse-transcribes an integral RNA template to add short DNA repeats to the 39-ends of telomeres. G-quadruplex structure in a DNA substrate can block its extension by telomerase. We have found that hnRNP A1-which was previously implicated in telomere length regulation-binds to both single-stranded and structured human telomeric repeats, and in the latter case, it disrupts their higher-order structure. Using an in vitro telomerase assay, we observed that depletion of hnRNP A/B proteins from 293 human embryonic kidney cell extracts dramatically reduced telomerase activity, which was fully recovered upon addition of purified recombinant hnRNP A1. This finding suggests that hnRNP A1 functions as an auxiliary, if not essential, factor of telomerase holoenzyme. We further show, using chromatin immunoprecipitation, that hnRNP A1 associates with human telomeres in vivo. We propose that hnRNP A1 stimulates telomere elongation through unwinding of a G-quadruplex or G-G hairpin structure formed at each translocation step.

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The RNA-binding domain of the protein kinase DAI, the double-stranded RNA inhibitor of translation, contains two repeats of a motif that is also found in a number of other RNA-binding proteins. This motif consists of 67 amino acid residues and is predicted to contain a positively charged alpha helix at its C terminus. We have analyzed the effects of equivalent single amino acid changes in three conserved residues distributed over each copy of the motif. Mutants in the C-terminal portion of either repeat were severely defective, indicating that both copies of the motif are essential for RNA binding. Changes in the N-terminal and central parts of the motif were more debilitating if they were made in the first motif than in the second, suggesting that the first motif is the more important for RNA binding and that the second motif is structurally more flexible. When the second motif was replaced by a duplicate of the first motif, the ectopic copy retained its greater sensitivity to mutation, implying that the two motifs have distinct functions with respect to the process of RNA binding. Furthermore, the mutations have the same effect on the binding of double-stranded RNA and VA RNA, consistent with the existence of a single RNA-binding domain for both activating and inhibitory RNAs.

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.

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