Genetic analysis of functional domains within the Drosophila LARK RNA-binding protein

LARK is an essential Drosophila RNA-binding protein of the RNA recognition motif (RRM) class that functions during embryonic development and for the circadian regulation of adult eclosion. LARK protein contains three consensus RNA-binding domains: two RRM domains and a retroviral-type zinc finger (RTZF). To show that these three structural domains are required for function, we performed a site-directed mutagenesis of the protein. The analysis of various mutations, in vivo, indicates that the RRM domains and the RTZF are required for wild-type LARK functions. RRM1 and RRM2 are essential for viability, although interestingly either domain can suffice for this function. Remarkably, mutation of either RRM2 or the RTZF results in the same spectrum of phenotypes: mutants exhibit reduced viability, abnormal wing and mechanosensory bristle morphology, female sterility, and flightlessness. The severity of these phenotypes is similar in single mutants and double RRM2; RTZF mutants, indicating a lack of additivity for the mutations and suggesting that RRM2 and the RTZF act together, in vivo, to determine LARK function. Finally, we show that mutations in RRM1, RRM2, or the RTZF do not affect the circadian regulation of eclosion, and we discuss possible interpretations of these results. This genetic analysis demonstrates that each of the LARK structural domains functions in vivo and indicates a pleiotropic requirement for both the LARK RRM2 and RTZF domains.     

Multiple protein functions of paired in Drosophila development and their conservation in the Gooseberry and Pax3 homologs

The Drosophila segmentation gene paired, whose product is homologous to the Drosophila Gooseberry and mammalian Pax3 proteins, has three general functions: proper development of the larval cuticle, survival to adulthood and male fertility. Both DNA-binding domains, the conserved N-terminal paired-domain and prd-type homeodomain, are required within the same molecule for all general paired functions, whereas a conserved His-Pro repeat located near its C terminus is a transactivation domain potentiating these functions. The C-terminal moiety of Paired includes two additional functional motifs: one, also present in Gooseberry and Pax3, is required for segmentation and cuticle development; the other, retained only in Gooseberry, is necessary for survival. The male fertility function, which cannot be replaced by Gooseberry and Pax3, is specified by the conserved N-terminal rather than the divergent C-terminal moiety of Paired. We conclude that the functional diversification of paired, gooseberry and Pax3, primarily determined by variations in their enhancers, is modified by adaptations of their coding regions as a necessary consequence of their newly acquired spatiotemporal expression.     

Functional Domains of the Rsp5 Ubiquitin-Protein Ligase

RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase. Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.     

Structure-function analysis of the Drosophila retinal determination protein Dachshund

Dachshund (Dac) is a highly conserved nuclear protein that is distantly related to the Ski/Sno family of corepressor proteins. In Drosophila, Dac is necessary and sufficient for eye development and, along with Eyeless (Ey), Sine oculis (So), and Eyes absent (Eya), forms the core of the retinal determination (RD) network. In vivo and in vitro experiments suggest that members of the RD network function together in one or more complexes to regulate the expression of downstream targets. For example, Dac and Eya synergize in vivo to induce ectopic eye formation and they physically interact through conserved domains. Dac contains two highly conserved domains, named DD1 and DD2, but no function has been assigned to either of them in an in vivo context. We performed structure-function studies to understand the relationship between the conserved domains of Dac and the rest of the protein and to determine the function of each domain during development. We show that only DD1 is essential for Dac function and while DD2 facilitates DD1, it is not absolutely essential in spite of more than 500 million years of conservation. Moreover, the physical interaction between Eya and DD2 is not required for the genetic synergy between the two proteins. Finally, we show that DD1 also plays a central role for nuclear localization of Dac.     

Mechanism of activation of the double-stranded-RNA-dependent protein kinase, PKR: role of dimerization and cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (eIF2).

An important defense against viral infection involves inhibition of translation by PKR phosphorylation of the alpha subunit of eIF2. Binding of viral dsRNAs to two dsRNA-binding domains (dsRBDs) in PKR leads to relief of an inhibitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggesting that these sequences carry out additional functions required for PKR control. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and assayed the chimeras for in vivo activity. We found that fusion of a heterologous dimerization domain with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ribosome association and we proposed that such targeting increases the localized concentration of PKR, enhancing interaction between PKR molecules. We addressed this premise by linking the truncated PKR to RAS sequences mediating farnesylation and membrane localization and found that the fusion protein was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Alanine substitution for the phosphorylation site, threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins, while aspartate or glutamate substitutions partially restored the function of the truncated kinase. These results indicate that both dimerization and cellular localization play a role in transient protein-protein interactions and that trans-autophosphorylation is the final step in the mechanism of activation of PKR.     

Structural requirements for notch signalling with delta and serrate during the development and patterning of the wing disc of Drosophila

The delta and Serrate proteins interact with the extracellular domain of the Notch receptor and initiate signalling through the receptor. The two ligands are very similar in structure and have been shown to be interchangeable experimentally; however, loss of function analysis indicates that they have different functions during development and analysis of their signalling during wing development indicates that the Fringe protein can discriminate between the two ligands. This raises the possibility that the signalling of delta and Serrate through Notch requires different domains of the Notch protein. Here we have tested this possibility by examining the ability of delta and Serrate to interact and signal with Notch molecules in which different domains had been deleted. This analysis has shown that EGF-like repeats 11 and 12, the RAM-23 and cdc10/ankyrin repeats and the region C-terminal to the cdc10/ankyrin repeats of Notch are necessary for both delta and Serrate to signal via Notch. They also indicate, however, that delta and Serrate utilise EGF-like repeats 24-26 of Notch for signalling, but there are significant differences in the way they utilise these repeats.     

Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.     

Roles of Oxa1-related inner-membrane translocases in assembly of respiratory chain complexes

Members of the family of the polytopic inner membrane proteins are related to Saccharomyces cerevisiae Oxa1 function in the assembly of energy transducing complexes of mitochondria and chloroplasts. Here we focus on the two mitochondrial members of this family, Oxa1 and Cox18, reviewing studies on their biogenesis as well as their functions, reflected in the phenotypic consequences of their absence in various organisms. In yeast, cytochrome c oxidase subunit II (Cox2) is a key substrate of these proteins. Oxa1 is required for co-translational translocation and insertion of Cox2, while Cox18 is necessary for the export of its C-terminal domain. Genetic and biochemical strategies have been used to investigate the functions of distinct domains of Oxa1 and to identify its partners in protein insertion/translocation. Recent work on the related bacterial protein YidC strongly indicates that it is capable of functioning alone as a translocase for hydrophilic domains and an insertase for TM domains. Thus, the Oxa1 and Cox18 probably catalyze these reactions directly in a co- and/or posttranslational way. In various species, Oxa1 appears to assist in the assembly of different substrate proteins, although it is still unclear how Oxa1 recognizes its substrates, and whether additional factors participate in this beyond its direct interaction with mitochondrial ribosomes, demonstrated in S. cerevisiae. Oxa1 is capable of assisting posttranslational insertion and translocation in isolated mitochondria, and Cox18 may posttranslationally translocate its only known substrate, the Cox2 C-terminal domain, in vivo. Detailed understanding of the mechanisms of action of these two proteins must await the resolution of their structure in the membrane and the development of a true in vitro mitochondrial translation system.     

RS domains contact the pre-mRNA throughout spliceosome assembly

SR proteins are essential metazoan splicing factors that contain an RNA-binding domain and an arginine/serine-rich domain that functions to promote assembly of the spliceosome. The prevailing model over the past several years suggests that the RS domains function as protein-interaction domains. However, two new papers from Green et al. demonstrate that these RS domains directly contact the pre-mRNA within the functional spliceosome. The sequential character of these contacts suggests that RS domain interactions with RNA promote spliceosome assembly.     

Regulators of G protein signaling: a bestiary of modular protein binding domains

Members of the newly discovered regulator of G protein signaling (RGS) families of proteins have a common RGS domain. This RGS domain is necessary for conferring upon RGS proteins the capacity to regulate negatively a variety of Galpha protein subunits. However, RGS proteins are more than simply negative regulators of signaling. RGS proteins can function as effector antagonists, and recent evidence suggests that RGS proteins can have positive effects on signaling as well. Many RGS proteins possess additional C- and N-terminal modular protein-binding domains and motifs. The presence of these additional modules within the RGS proteins provides for multiple novel regulatory interactions performed by these molecules. These regions are involved in conferring regulatory selectivity to specific Galpha-coupled signaling pathways, enhancing the efficacy of the RGS domain, and the translocation or targeting of RGS proteins to intracellular membranes. In other instances, these domains are involved in cross-talk between different Galpha-coupled signaling pathways and, in some cases, likely serve to integrate small GTPases with these G protein signaling pathways. This review discusses these C- and N-terminal domains and their roles in the biology of the brain-enriched RGS proteins. Methods that can be used to investigate the function of these domains are also discussed.