Hedgehog (Hh) proteins play diverse organizing roles in development by regulating gene expression in responding cells. The Gli homolog Cubitus interruptus (Ci) is involved in controlling the transcription of Hh target genes. A repressor form of Ci arises in the absence of Hh signaling by proteolytic cleavage of intact Ci. We show that this cleavage is essential for limb patterning and is regulated by Hh in vivo. We provide evidence for the existence of a distinct activator form of Ci, which does not arise by mere prevention of Ci proteolysis, but rather depends on a separate regulatory step subject to Hh control. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. Thus, limb development is organized by the integration of different transcriptional outputs of Hh signaling.

Eukaryotic translation initiation factor‐4A (eIF‐4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA‐dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA‐ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF‐4F. To gain further understanding into the mechanism of action of eIF‐4A in mRNA‐ribosome binding, defective eIF‐4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF‐4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap‐independent internal initiation mechanism. Addition of eIF‐4A or eIF‐4F relieved inhibition of translation, but eIF‐4F was six times more effective than eIF‐4A, whereas eIF‐4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF‐4A is defective in recycling through eIF‐4F, thus explaining the dramatic inhibition of translation. Mutant eIF‐4A proteins also inhibited eIF‐4F‐dependent, but not eIF‐4A‐dependent RNA helicase activity. Taken together these results suggest that eIF‐4A functions primarily as a subunit of eIF‐4F, and that singular eIF‐4A is required to recycle through the complex during translation. Surprisingly, eIF‐4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.

eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eEF-4B, it exhibits duplex RNA helicase activity. eEF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of elIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduce eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity.Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B.Binding of 40S ribosomes to mRNA in eukaryotes is an intricate process that requires the participation of at least three eukaryotic initiation factors, eIF-4A, eIF-4B, and eIF-4F, and the hydrolysis of ATP (for reviews, see references 35, 45, and 53). eIF-4A is a 50-kDa polypeptide that exhibits single-stranded RNA-dependent ATPase activity and, in combination with eIF-4B, duplex RNA helicase activity (17,44,46). There are two eIF-4A gene products, eIF-4AI and eIF-4AII, that are almost identical throughout the coding sequence except for the amino-terminal 20 amino acids (39). Both forms exchange into the eIF-4F complex (60). eIF-4F is a three-subunit complex (12, 19, 56) comprising (i) eIF-4E, a 24-kDa polypeptide that contains the cap-binding site (54); (ii) eIF-4A, which is a mixture of eIF-4AI and eIF-4AII (4:1) (9); and (iii) p220, a 220-kDa polypeptide whose function is not clear but whose integrity is apparently important for eIF-4F function (13). It is t...

The interferon-inducible, double-stranded RNA-dependent protein kinase PKR has been implicated in anti-viral, anti-tumor, and apoptotic responses. Others have attempted to examine the requirement of PKR in these roles by targeted disruption at the amino terminal-encoding region of the Pkr gene. By using a strategy that aims at disruption of the catalytic domain of PKR, we have generated mice that are genetically ablated for functional PKR. Similar to the other mouse model of Pkr disruption, we have observed no consequences of loss of PKR on tumor suppression. Anti-viral response to influenza and vaccinia also appeared to be normal in mice and in cells lacking PKR. Cytokine signaling in the type I interferon pathway is normal but may be compromised in the erythropoietin pathway in erythroid bone marrow precursors. Contrary to the aminoterminal targeted Pkr mouse, tumor necrosis factor ␣-induced apoptosis and the anti-viral apoptosis response to influenza is not impaired in catalytic domain-targeted Pkrnull cells. The observation of intact eukaryotic initiation factor-2␣ phosphorylation in these Pkr-null cells provides proof of rescue by another eukaryotic initiation factor-2␣ kinase(s).

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.