Prevention of cross-talk in conserved regulatory systems: identification of specificity determinants in RNA-binding anti-termination proteins of the BglG family

Each family of signal transduction systems requires specificity determinants that link individual signals to the correct regulatory output. In Bacillus subtilis, a family of four anti-terminator proteins controls the expression of genes for the utilisation of alternative sugars. These regulatory systems contain the anti-terminator proteins and a RNA structure, the RNA anti-terminator (RAT) that is bound by the anti-terminator proteins. We have studied three of these proteins (SacT, SacY, and LicT) to understand how they can transmit a specific signal in spite of their strong structural homology. A screen for random mutations that render SacT capable to bind a RNA structure recognized by LicT only revealed a substitution (P26S) at one of the few non-conserved residues that are in contact with the RNA. We have randomly modified this position in SacT together with another non-conserved RNA-contacting residue (Q31). Surprisingly, the mutant proteins could bind all RAT structures that are present in B. subtilis. In a complementary approach, reciprocal amino acid exchanges have been introduced in LicT and SacY at non-conserved positions of the RNA-binding site. This analysis revealed the key role of an arginine side-chain for both the high affinity and specificity of LicT for its cognate RAT. Introduction of this Arg at the equivalent position of SacY (A26) increased the RNA binding in vitro but also resulted in a relaxed specificity. Altogether our results suggest that this family of anti-termination proteins has evolved to reach a compromise between RNA binding efficacy and specific interaction with individual target sequences.     

The RGK family: a regulatory tail of small GTP-binding proteins

RGK proteins are small Ras-related GTP-binding proteins that function as potent inhibitors of voltage-dependent calcium channels, and two members of the family, Gem and Rad, modulate Rho-dependent remodeling of the cytoskeleton. Within the Ras superfamily, RGK proteins have distinct structural and regulatory characteristics. It is an open question as to whether RGK proteins catalyze GTP hydrolysis in vivo. Binding of calmodulin and the 14-3-3 protein to RGK proteins controls downstream pathways. Here, we discuss the structural and functional properties of RGK proteins and highlight recent work by Beguin and colleagues addressing the mechanism of Gem regulation by calmodulin and 14-3-3.     

Genomic definition of RIM proteins: evolutionary amplification of a family of synaptic regulatory proteins

RIMs are synaptic proteins that are essential for normal neurotransmitter release. We now show that while invertebrates contain only a single RIM gene, vertebrates contain four: two large genes encoding RIM1alpha (0.50 Mb) or RIM2alpha, 2beta, and 2gamma (0.50-0.75 Mb) and two smaller genes encoding RIM3gamma (14 kb) or RIM4gamma (55 kb). RIM1alpha and RIM2alpha consist of an N-terminal Zn(2+)-finger domain, central PDZ and C(2)A domains, and a C-terminal C(2)B domain; RIM2beta consists of a short beta-specific sequence followed by central PDZ and C(2)A domains and a C-terminal C(2)B domain; and RIM2gamma, 3gamma, and 4gamma consist of only a C(2)B domain. In the RIM2 gene, RIM2beta and 2gamma are transcribed from internal promoters. alpha- and beta-RIMs are extensively alternatively spliced at three canonical positions, resulting in >200 variants that differ by up to 400 residues. Thus gene duplication, alternative splicing, and multiple promoters diversify a single invertebrate RIM into a large vertebrate protein family. The multiplicity of vertebrate RIMs may serve to fine-tune neurotransmitter release beyond a fundamental, evolutionarily conserved, and common function for RIMs.     

Tribbles: a family of kinase-like proteins with potent signalling regulatory function

The recent identification of tribbles as regulators of signal processing systems and physiological processes, including development, together with their potential involvement in diabetes and cancer, has generated considerable interest in these proteins. Tribbles have been reported to regulate activation of a number of intracellular signalling pathways with roles extending from mitosis and cell activation to apoptosis and modulation of gene expression. The current review summarises our current understanding of interactions between tribbles and various other proteins. Since our understanding on the molecular basis of tribbles function is far from complete, we also describe a bioinformatic analysis of various segments of tribbles proteins, which has revealed a number of highly conserved peptide motifs with potentially important functional roles.     

Identification of bacterial sRNA regulatory targets using ribosome profiling

Bacteria express large numbers of non-coding, regulatory RNAs known as ‘small RNAs’ (sRNAs). sRNAs typically regulate expression of multiple target messenger RNAs (mRNAs) through base-pairing interactions. sRNA:mRNA base-pairing often results in altered mRNA stability and/or altered translation initiation. Computational identification of sRNA targets is challenging due to the requirement for only short regions of base-pairing that can accommodate mismatches. Experimental approaches have been applied to identify sRNA targets on a genomic scale, but these focus only on those targets regulated at the level of mRNA stability. Here, we utilize ribosome profiling (Ribo-seq) to experimentally identify regulatory targets of the Escherichia coli sRNA RyhB. We not only validate a majority of known RyhB targets using the Ribo-seq approach, but also discover many novel ones. We further confirm regulation of a selection of known and novel targets using targeted reporter assays. By mutating nucleotides in the mRNA of a newly discovered target, we demonstrate direct regulation of this target by RyhB. Moreover, we show that Ribo-seq distinguishes between mRNAs regulated at the level of RNA stability and those regulated at the level of translation. Thus, Ribo-seq represents a powerful approach for genome-scale identification of sRNA targets.     

Possible function of RNA-binding proteins of eukaryotes: blocking of RNA accessibility for DNA-interacting (regulatory) protein

In the presence of rat liver cytosol the glucocorticoid receptor complexes (GRC) bind very weakly to RNA as compared to DNA, whereas the binding of purified GRC to RNA is only 2-3 time less than to DNA. It is assumed that the RNA-binding proteins present in the cytoplasm selectively prevent the GRC binding to RNA and thus facilitate the GRC binding to nuclear DNA (chromatin). This blocking function may be performed by the RNA-binding proteins with respect to different regulatory DNA-dependent intracellular proteins in vivo. Possible mechanisms of regulation of the genome activity based on changes in RNA-RNA-binding proteins ratio in intact cells and in individual cell compartments are discussed.     

Shared RNA-binding sites for interacting members of the Drosophila ELAV family of neuronal proteins

The product of the Drosophila embryonic lethal abnormal visual system is a conserved protein (ELAV) necessary for normal neuronal differentiation and maintenance. It possesses three RNA-binding domains and is involved in the regulation of RNA metabolism. The long elav 3′-untranslated region (3′-UTR) is necessary for autoregulation. We used RNA-binding assays and in vitro selection to identify the ELAV best binding site in the elav 3′-UTR. This site resembles ELAV-binding sites identified previously in heterologous targets, both for its nucleotide sequence and its significant affinity for ELAV (K_d 40 nM). This finding supports our model that elav autoregulation depends upon direct interaction between ELAV and elav RNA. We narrowed down the best binding site to a 20 nt long sequence A(U₅)A(U₃)G(U₂)A(U₆) in an alternative 3′ exon. We propose and test a model in which the regulated use of this alternative 3′ exon is involved in normal elav regulation. Found in NEurons (FNE), another neuronal RNA-binding protein paralogous to ELAV, also binds this site. These observations provide a molecular basis for the in vivo interactions reported previously between elav and fne.     

Bul proteins, a nonredundant, antagonistic family of ubiquitin ligase regulatory proteins

Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination.