Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Interaction of Actin and the Chloroplast Protein Import Apparatus

Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.Actin is a ubiquitous protein of eukaryotic cells. Actin microfilaments are formed from polymerization of actin monomers and are a major component of the cytoskeleton. In plant cells, actin microfilaments are arranged in longitudinal arrays of thick actin bundles with randomly oriented thin actin filaments extending from the bundles (1). Chloroplasts are either aligned along the actin bundles or closely associated with the fine filaments and are surrounded by baskets of actin microfilaments (1, 2). A direct interaction of chloroplasts with the actin cytoskeleton has been postulated to anchor chloroplasts at appropriate intracellular positions (3). Chloroplast movement depends on cytosolic actin filaments and is stimulated by high light intensity (4). A chloroplast envelope protein involved in blue light-dependent chloroplast repositioning has been identified by the analysis of the Arabidopsis chup1 (chloroplast unusual positioning 1) mutant, which was unable to relocate its chloroplasts under high light stimulation (5). CHUP1 is a protein exclusively targeted to the chloroplast outer envelope membrane that is essential for chloroplast anchorage to the plasma membrane (6). CHUP1 interacts with actin and profilin, a modulator of actin polymerization, and it may play a regulatory role in actin polymerization during chloroplast photo-relocation (7).The interaction of amyloplasts with the actin cytoskeleton has been implicated in gravity perception and signal transduction. Several models for the role of the actin cytoskeleton have been proposed (8), but the nature of the interaction is not known. However, disruption of the actin cytoskeleton enhanced sedimentation of amyloplasts and promoted gravitropism (9, 10), and a role for myosin has been proposed on the basis of inhibitor experiments (11).The actin cytoskeleton and myosin have also been implicated in plastid stromule movement. Stromules (stroma-filled tubules) are highly dynamic tubular structures extending from the surface of all plastid types (12, 13). Stromules are delimited by the inner and outer plastid envelope membranes, which are closely associated (for a review, see Refs. 12 and 13). Experiments with inhibitors of microfilament- and microtubule-based movement suggested that stromules move along actin microfilaments powered by the ATPase activity of myosin motors (14). Physical connection between the envelope membranes seems likely to be required to provide a means of coordinating the movement of the inner envelope membrane with the microfilament-associated outer envelope membrane. There is evidence for direct connection of the inner and outer envelope membranes at contact sites, which support protein translocation through the protein import apparatus (15, 16). This apparatus consists of two membrane protein complexes that associate to allow translocation of nucleus-encoded proteins from the cytoplasm to the interior stromal compartment (for a review, see 17). The translocon at the outer envelope membrane of chloroplasts (TOC complex)² mediates the initial recognition of preproteins and their translocation across the outer membrane (18). The translocon at the inner envelope membrane of chloroplasts (TIC complex) physically associates with the TOC complex and provides the membrane translocation channel for the inner membrane. In addition, the TOC and TIC complexes interact with a set of molecular chaperones, which assist the transfer of imported proteins (19–21).With the aim of identifying components involved in the interaction of the chloroplast envelope with the actin cytoskeleton, we have used actin co-immunoprecipitation and co-sedimentation experiments with detergent-solubilized pea chloroplast envelope membranes. Components of the TOC-TIC protein import apparatus have been identified by mass spectrometry and Western blotting, and a direct interaction of Escherichia coli-expressed Toc159 with actin was demonstrated by co-sedimentation. This interaction may have a so far unrecognized physiological role in chloroplast protein import.