Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.     

SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.     

Identification of a novel human DDX40 gene, a new member of the DEAH-box protein family

 The DExH/D-box superfamily of RNA helicases seems to play key roles during RNA metabolism, such as pre-mRNA splicing, ribosome biogenesis, and others. We have cloned a new gene of the DEAH-box protein subgroup, designated DDX40 (DEAD/H-box polypeptide 40 gene). DDX40 contains 3656 nucleotides and codes for a putative 779-amino-acid protein. Sequence analysis of the cDNA product revealed that it contained a DEAH (Asp-Glu-Ala-His) sequence motif and other conserved motifs. The DDX40 protein shared 53% and 43% amino acid identity with human DDX8 and yeast Drh1, respectively, in the conserved region. Northern blot analysis showed that DDX40 was expressed ubiquitously in the eight tissues examined, implying a general physiological function of the protein. We speculate that, like other members of the DExH/D-box superfamily, DDX40 may play roles in pre-mRNA splicing, ribosome biogenesis and other RNA processing functions. Received: August 7, 2002 / Accepted: September 12, 2002     

ABC-F proteins in mRNA translation and antibiotic resistance

The ATP binding cassette protein superfamily comprises ATPase enzymes which are, for the most part, involved in transmembrane transport. Within this superfamily however, some protein families have other functions unrelated to transport. One example is the ABC-F family, which comprises an extremely diverse set of cytoplasmic proteins. All of the proteins in the ABC-F family characterized to date act on the ribosome and are translation factors. Their common function is ATP-dependent modulation of the stereochemistry of the peptidyl transferase center (PTC) in the ribosome coupled to changes in its global conformation and P-site tRNA binding geometry. In this review, we give an overview of the function, structure, and theories for the mechanisms-of-action of microbial proteins in the ABC-F family, including those involved in mediating resistance to ribosome-binding antibiotics.     

Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged

Alternative splicing of mRNA precursors is a nearly ubiquitous and extremely flexible point of gene control in humans. It provides cells with the opportunity to create protein isoforms of differing, even opposing, functions from a single gene. Cancer cells often take advantage of this flexibility to produce proteins that promote growth and survival. Many of the isoforms produced in this manner are developmentally regulated and are preferentially re-expressed in tumors. Emerging insights into this process indicate that pathways that are frequently deregulated in cancer often play important roles in promoting aberrant splicing, which in turn contributes to all aspects of tumor biology.     

The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1

Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.     

A role for VICKZ proteins in the progression of colorectal carcinomas: regulating lamellipodia formation

VICKZ proteins are a highly conserved family of RNA binding proteins, implicated in RNA regulatory processes such as intracellular RNA localization, RNA stability, and translational control. During embryogenesis, VICKZ proteins are required for neural crest migration and in adults, the proteins are overexpressed primarily in different cancers. We hypothesized that VICKZ proteins may play a role in cancer cell migration. In patients, VICKZ expression varies with tumour type, with over 60% of colon, lung, and ovarian tumours showing strong expression. In colorectal carcinomas (CRCs), expression is detected at early stages, and the frequency and intensity of staining increase with progression of the disease to lymph node metastases, of which 97% express the protein at high levels. Indeed, in stage II CRC, the level of VICKZ expression in the primary lesion correlates with the degree of lymph node metastasis. In culture, VICKZ proteins rapidly accumulate in processes at the leading edge of PMA-stimulated SW480 CRC cells, where they co-localize with beta-actin mRNA. Two distinct cocktails of shRNAs, each targeting all three VICKZ paralogues, cause a dramatic drop in lamellipodia and ruffle formation in stimulated cells. Thus, VICKZ proteins help to facilitate the dynamic cell surface morphology required for cell motility. We propose that these proteins play an important role in CRC metastasis by shuttling requisite RNAs to the lamellipodia of migrating cells.     

Regulation of the Dbp5 ATPase cycle in mRNP remodeling at the nuclear pore: a lively new paradigm for DEAD-box proteins

It is commonly assumed that all DEAD-box ATPases function via a shared mechanism, since this is the case for the few proteins characterized thus far. Hodge and colleagues (pp. 1052-1064) and Noble and colleagues (pp. 1065-1077) now describe a novel model for Dbp5's ATPase cycle in mRNA (messenger RNA)/protein complex (mRNP) remodeling during nuclear export. Notably, unlike other DEAD-box proteins, Dbp5 uses a conformational change distinct from ATP hydrolysis for its activity and requires an ADP release factor to reset its ATPase cycle.     

A hypothetical new role for single-stranded DNA binding proteins in the immune system

The breadth of the host range of single-stranded DNA (ssDNA) viruses is roughly comparable to the host range of double-stranded DNA viruses (dsDNA). Yet, general ssDNA sensing receptors that activate the immune system have not been unequivocally identified while numerous dsDNA sensing receptors are known. Here, we hypothesize that some of the Single-Stranded DNA Binding (SSB) proteins may act as receptors that detect single-stranded DNA from pathogens and activate the innate immune system. As the first test of our hypothesis, we checked whether human genes that are known to bind to ssDNA are potentially interferon-regulated. Out of the 102 human genes that are known to have ssDNA binding ability 23 genes show a more than two-fold increase in gene expression upon interferon treatment. Single-stranded DNA viruses are pathogens of not only animals but also of plants and protozoans. We used this information to further prioritize our candidate list to ssDNA binding genes that are common between the model plant Arabidopsis thaliana and humans. Based on these strategies, we shortlist several promising candidate genes including the HMGB1 gene which could act as a ssDNA sensor that activates the immune system. Agreeably though we cannot establish a definitive role for these genes as ssDNA sensors of the immune system as yet, our preliminary analysis suggests the potential existence of ssDNA binding protein-like receptors (SLR's) that are worth investigating further.     

Novel proteomic approach (PUNCH-P) reveals cell cycle-specific fluctuations in mRNA translation

Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions.     

Influenza virus polymerase confers independence of the cellular cap-binding factor eIF4E for viral mRNA translation

The influenza virus mRNAs are structurally similar to cellular mRNAs nevertheless; the virus promotes selective translation of viral mRNAs despite the inhibition of host cell protein synthesis. The infection proceeds normally upon functional impairment of eIF4E cap-binding protein, but requires functional eIF4A helicase and eIF4G factor. Here, we have studied whether the presence of cis elements in viral mRNAs or the action of viral proteins is responsible for this eIF4E-independence. The eIF4E protein is required for viral mRNA translation in vitro, indicating that cis-acting RNA sequences are not involved in this process. We also show that PB2 viral polymerase subunit interacts with the eIF4G protein. In addition, a chimeric mRNA containing viral UTR sequences transcribed by the viral polymerase out of the infection is successfully translated independently of an impaired eIF4E factor. These data support that the viral polymerase is responsible for the eIF4E independence of influenza virus mRNA translation.     

The importance of inter- and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mRNA

Calicivirus structure proteins are expressed from a subgenomic mRNA with two overlapping cistrons. The first ORF of this RNA codes for the viral major capsid protein VP1, and the second for the minor capsid protein VP2. Translation of VP2 is mediated by a termination/reinitiation mechanism, which depends on an upstream sequence element of ~70 nucleotides denoted “termination upstream ribosomal binding site” (TURBS). Two short sequence motifs within the TURBS were found to be essential for reinitiation. By a whole set of single site mutations and reciprocal base exchanges we demonstrate here for the first time conclusive evidence for the necessity of mRNA/18S rRNA hybridization for translation reinitiation in an eukaryotic system. Moreover, we show that motif 2 exhibits intramolecular hybridization with a complementary region upstream of motif 1, thus forming a secondary structure that positions post-termination ribosomes in an optimal distance to the VP2 start codon. Analysis of the essential elements of the TURBS led to a better understanding of the requirements for translation termination/reinitiation in eukaryotes.     

The cannabinoid antagonist SR141716A facilitates memory acquisition and consolidation in the mouse elevated T-maze

Δ⁹-THC and synthetic cannabinoids produce memory impairment in humans as well as in laboratory animals. The high concentration of cannabinoid CB1 receptors and the presence of endocannabinoids in the hippocampus suggest that a cannabinoid neurochemical system may play a role in learning and memory processes. Thus, the objective of the present work was to study the effect of the cannabinoid antagonist SR141716A (SR) on memory acquisition, consolidation and retrieval in a recently developed elevated T-maze (ETM) model of anxiety and memory. In addition, we investigated whether pre-training SR administration was capable of reversing scopolamine-induced memory impairment. Adult male mice were exposed to the closed arm as many times as necessary for the animals to reach the avoidance criterion of remaining in the closed arm for 300 s; they were then tested (exposed to the closed arm) 24 h and 7 days after the training. SR (0.5, 1.0 or 2.0 mg/kg) was administered i.p. 20 min before the training, immediately after training or 20 min before the test in the mice. The elevated plus-maze (EPM) was used to investigate a possible influence of SR on locomotion and on the anxiety-related behavior. SR provoked memory improvement, which was observed when the drug was administered before (effect on memory acquisition/consolidation) or immediately after the training (effect on memory consolidation), but not when the drug was administered before the test (effect on memory retrieval). Also, SR administration reversed scopolamine-induced amnesia. These effects were observed in the absence of changes in locomotion or anxiety levels. Our results demonstrate that the blockade of cannabinoid receptors may improve memory acquisition and consolidation in the ETM model.