MicroRNAs (miRNAs) are an emerging class of non-coding endogenous RNAs involved in multiple cellular processes, including cell differentiation. Treatment with retinoic acid (RA) results in neural differentiation of neuroblastoma cells. We wanted to elucidate whether miRNAs contribute to the gene expression changes induced by RA in neuroblastoma cells and whether miRNA regulation is involved in the transduction of the RA signal. We show here that RA treatment of SH-SY5Y neuroblastoma cells results in profound changes in the expression pattern of miRNAs. Up to 42 different miRNA species significantly changed their expression (26 up-regulated and 16 down-regulated). Among them, the closely related miR-10a and -10b showed the most prominent expression changes. Induction of miR-10a and -10b by RA also could be detected in LA-N-1 neuroblastoma cells. Loss of function experiments demonstrated that miR-10a and -10b are essential mediators of RA-induced neuroblastoma differentiation and of the associated changes in migration, invasion, and in vivo metastasis. In addition, we found that the SR-family splicing factor SFRS1 (SF2/ASF) is a target for miR-10a -and -10b in HeLa and SH-SY5Y neuroblastoma cells. We show here that changes in miR-10a and -10b expression levels may regulate SFRS1-dependent alternative splicing and translational functions. Taken together, our results give support to the idea that miRNA regulation plays a key role in RA-induced neuroblastoma cell differentiation. The discovery of SFRS1 as direct target of miR-10a and -10b supports the emerging functional interaction between two post-transcriptional mechanisms, microRNAs and splicing, in the neuronal differentiation context.

The insulin receptor (IR) exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. This alternative splicing is cell specific, and the relative proportions of exon 11 isoforms also vary during development, aging, and different disease states. We have previously demonstrated that both intron 10 and exon 11 contain regulatory sequences that affect IR splicing both positively and negatively. In this study, we sought to define the precise sequence elements within exon 11 that control exon recognition and cellular factors that recognize these elements. Using minigenes carrying linker-scanning mutations within exon 11, we detected both exonic splicing enhancer and exonic splicing silencer elements. We identified binding of SRp20 and SF2/ASF to the exonic enhancers and CUG-BP1 to the exonic silencer by RNA affinity chromatography. Overexpression and knockdown studies with hepatoma and embryonic kidney cells demonstrated that SRp20 and SF2/ASF increase exon inclusion but that CUG-BP1 causes exon skipping. We found that CUG-BP1 also binds to an additional intronic splicing silencer, located at the 3' end of intron 10, to promote exon 11 skipping. Thus, we propose that SRp20, SF2/ASF, and CUG-BP1 act antagonistically to regulate IR alternative splicing in vivo and that the relative ratios of SRp20 and SF2/ASF to CUG-BP1 in different cells determine the degree of exon inclusion.

To elucidate how methylation of specific sites in plant DNA might control transcription, we examined the effect of DNA methylation at CpG sequences on the binding of plant nuclear factors to an oligonucleotide duplex containing the consensus sequence for mammalian CREB (cAMP response element binding protein). CREB is part of the ATF (activating transcription factor) family of mammalian proteins specifically binding to 5'-TGACGTCA-3' and related sequences. Proteins recognizing the CREB-specific ligand were identified in nuclear extracts of pea seeds, wheat germ, cauliflower, and soybean leaves using electrophoretic mobility shift assays. Cytosine methylation inhibited binding of this protein in all these extracts, and so this sequence-specific DNA-binding activity is referred to as methylation-inhibited binding protein 1 (MIB-1). Sites somewhat similar to that of the CREB ligand are found in the upstream regions of a wheat histone H3 gene and tomato and pea ribulose 1,5-bisphosphate carboxylase genes. These sites were bound preferentially by distinct proteins that may be related to the previously described plant proteins HBP-1, HSBF, ASF-1, or GBF. Methylation of cytosine residues at these sites and at a site for MIB-1 located upstream of a soybean proline-rich protein gene also reduced specific binding with all the nuclear extracts tested. Similarly, substitution of the central CpG dinucleotide with TpG decreased binding.

Pre-mRNA splicing requires a large number of RNA-binding proteins that have one or more RNA-recognition motifs (RRMs). Among these is the SR protein family, whose members are essential for splicing and are able to commit pre-mRNAs to the splicing pathway with overlapping but distinct substrate specificity. Some SR proteins, such as SC35, contain an N-terminal RRM and a C-terminal arginine/serine-rich (RS) domain, whereas others, such as SF2/ASF, also contain a second, atypical RRM. Although both the RRMs and the RS domain of SR proteins are required for constitutive splicing, it is unclear which domain(s) defines their substrate specificity, and whether two RRMs in a given SR protein function independently or act coordinately. Using domain swaps between SC35 and SF2/ASF and a functional commitment assay, we demonstrate that individual domains are functional modules, RS domains are interchangeable, and substrate specificity is defined by the RRMs. The atypical RRM of SF2/ASF does not appear to function alone in splicing, but can either activate or suppress the splicing specificity of an N-terminal RRM. Therefore, multiple RRMs in SR proteins act coordinately to achieve a unique spectrum of pre-mRNA substrate specificity.

SR-protein-specific kinase 1 (SRPK1) is first identified as a specific kinase for SR splicing factors. By RT-PCR of a conserved kinase domain, novel SR-protein-specific kinase clones were isolated from mouse brain. The cloned cDNAs encode a 106 kDa protein (648 amino acids, 92% identical to human SRPK1) and a 120 kDa protein (681 amino acids, 58% identical to human SRPK1). Therefore, they were designated mSRPK1 and mSRPK2, respectively. Northern blotting revealed the ubiquitous expression of mSRPK1 in all tissues examined and the tissue-specific expression of mSRPK2 in testis, lung, and brain. Both kinases phosphorylated SF2/ASF, a member of SR proteins in vitro and the phosphopeptide mappings were identical, indicating that these kinases phosphorylate the same site of SF2/ASF. Overexpression of mSRPK2 caused disassembly of cotransfected SF2/ASF and endogenous SC35. Our results indicate that SRPK family members may regulate the disassembly of the SR proteins in a tissue-specific manner.

The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5' splice sites and of one major or one minor 3' splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5' splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5' splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3' splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5' splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

The airway provides numerous defense mechanisms to prevent microbial colonization by the large numbers of bacteria and viruses present in ambient air. An important component of this defense is the antimicrobial peptides and proteins present in the airway surface fluid (ASF), the mucin-rich fluid covering the respiratory epithelium. These include larger proteins such as lysozyme and lactoferrin, as well as the cationic defensin and cathelicidin peptides. While some of these peptides, such as human beta-defensin (hBD)-1, are present constitutively, others, including hBD2 and -3 are inducible in response to bacterial recognition by Toll-like receptor-mediated pathways. These peptides can act as microbicides in the ASF, but also exhibit other activities, including potent chemotactic activity for cells of the innate and adaptive immune systems, suggesting they play a complex role in the host defense of the airway. Inhibition of antimicrobial peptide activity or gene expression can result in increased susceptibility to infections. This has been observed with cystic fibrosis (CF), where the CF phenotype leads to reduced antimicrobial capacity of peptides in the airway. Pathogenic virulence factors can inhibit defensin gene expression, as can environmental factors such as air pollution. Such an interference can result in infections by airway-specific pathogens including Bordetella bronchiseptica, Mycobacterium tuberculosis, and influenza virus. Research into the modulation of peptide gene expression in animal models, as well as the optimization of peptide-based therapeutics shows promise for the treatment and prevention of airway infectious diseases.

Since the introduction of the virus into the Republic of Georgia in 2007 African swine fever (ASF) has become a large-scale epidemic involving the domestic pig population but wild boars are involved as well. From 2008 to 2009 the ASF epidemic affected wild and domestic pigs in all the southern regions of the Russian Federation (RF). The driving force of the epidemic in its initial stages was direct contact between infected wild boars and between wild boars and traditionally free-ranging domestic pigs in backyard farms. Driving forces of the epidemic at the its first stages was direct contact of infected wild boars between each other and with traditionally free ranged domestic pigs in backyard farms. The next stage developed due to illegal movement of pig products contaminated by African swine fever virus (ASFV) from affected regions and swill feeding, and inefficient implementation of measures to prevent and control ASF. From 2010 through 2012, ASF spread to other, previously unaffected regions of the RF. Most of outbreaks in the southern regions (Krasnodar, Stavropol, Rostov regions) are secondary. Currently, the disease situation observed in endemic areas of the RF, including the southern Krasnodar and Volgograd regions and the central Tver' region, is very complicated. In 2012, a large number of outbreaks in domestic pigs and in wild boars were reported. The circulating ASFV is highly virulent and has maintained its virulence throughout the epidemic since its introduction in 2007. Considering the forces currently driving the ASF epidemic - circulation of ASF virus in wild boars, ineffectiveness of prevention and control measures, lack of common interest in eradicating the disease and absence of a nationally funded eradication program - continued outbreaks, including those in previously unaffected regions of the RF, can be expected.

Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by homozygous inactivation of the SMN1 (Survival Motor Neuron 1) gene. The disease severity is mainly influenced by the copy number of SMN2, a nearly identical gene from which only low amounts of full-length mRNA are produced. This correlation is not absolute, suggesting the existence of yet unknown factors modulating disease progression. We identified and characterized the rare variant c.859G>C (p.Gly287Arg) in exon 7 in both SMN2 copies of a male patient affected with type III SMA, a milder form of the disease rarely associated with only two SMN2 copies. We demonstrated in vivo, in this patient and in a second unrelated patient, and ex vivo, using SMN splicing assays, that the variant induces inclusion of exon 7 into SMN2 mRNA. Moreover, we show that the c.859G>C variation is located in a composite splicing regulatory element in the centre of exon 7. The variation does not affect binding of HTra2â nor creates a novel SF2/ASF enhancer, but disrupts an hnRNP A1 binding site. The natural occurrence of enhanced inclusion of SMN2 exon 7 in milder SMA cases supports the current therapeutic strategies based on splicing modulation in SMA patients.

OBJECTIVE

Little is known about the relationship between current and past smoking behaviour and the severity of alcohol dependence. The purpose was to explore the strength of this relationship.

METHODS

A random population sample of 18 to 64 year-olds from northern Germany was used (n = 4075; participation rate: 70%). It included 761 cigarette smokers fulfilling at least one alcohol-dependence criterion. The severity of alcohol dependence according to the alcohol-dependence syndrome criteria frequency (ASF) was estimated by a standardized questionnaire based on diagnostic instruments of the alcohol dependence syndrome and which included five response categories, from 'never' to 'daily'. Nicotine dependence was diagnosed according to the Diagnostic and Statistical Manual of mental disorders (DSM-IV) with the Composite International Diagnostic Interview (CIDI). As a second measure, the Fagerström Test of Nicotine Dependence (FTND) was used.

RESULTS

The number of cigarettes and years of daily smoking, nicotine dependence, and the number of nicotine dependence symptoms each showed a relationship with the ASF. Effect size (w) were 0.17-0.21 for chi-squared (chi(2)) tests. In a general linear regression model with the ASF as the dependent variable (R(2) = 0.17), number of years of daily smoking, age at onset of smoking, number of attempts to reduce or quit, the number of nicotine-dependence symptoms according to DSM-IV and the FTND sum score were retained as independent variables.

CONCLUSIONS

Long-term smoking, a large number of nicotine-dependence symptoms according to DSM and a strong urge to smoke according to the FTND are related with a high ASF.

We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.

Between attacks, migraine with (MO) or without aura (MA) patients show deficient habituation of pattern-reversal visual evoked potentials (PR-VEP) and a strong intensity dependence of auditory evoked cortical potentials (IDAP). Clinical observations of migraine prodromes and previously published electrophysiological studies suggest that cortical information processing may vary in close temporal relationship to the attack. We studied PR-VEP and IDAP just before (11 MO pts), during (23 MO, 3 MA), 1 day following (27 MO, 1 MA) and 2 days following (14 MO) a migraine attack. The results were compared with a large group of MO patients recorded at a distance of at least 3 days from an attack (n = 66 for IDAP; n = 39 for VEP). Patients recorded the day before the attack had on average an habituation of -13.6+/-20.5% (mean +/- SD) between the 5th and 1st block of 100 averaged VEP responses and a flat (0.38+/-1.06 microV/10 dB) amplitude-stimulus intensity function (ASF) slope of the auditory evoked cortical potential. Both values were significantly different from those obtained in the attack interval (P=0.003; P=0.020). During the attack, VEP habituation was less pronounced (-0.17+/-26.2%) and ASF slopes remained flat (0.32+/-1.44 microV/10 dB; P=0.002 compared to interval). During the 2 days following the attack, VEP habituation was replaced by potentiation (+0.09+/-29.1% the 1st day; 19.5+/-45.7% the 2nd day) and ASF slopes increased markedly (0.87+/-1.39 and 1.14+/-1.12 microV/10 dB). The normalization of evoked cortical responses just before and during the attack, might reflect an increase in the cortical preactivation level due to enhanced activity in raphe-cortical serotonergic pathways.

A disease-causing G-to-T transversion at position +6 of BRCA1 exon 18 induces exclusion of the exon from the mRNA and, as has been suggested by in silico analysis, disrupts an ASF/SF2-dependent splicing enhancer. We show here using a pulldown assay with an internal standard that wild-type (WT) and mutant T6 sequences displayed similar ASF/SF2 binding efficiencies, which were significantly lower than that of a typical exonic splicing enhancer derived from the extra domain A exon of fibronectin. Overexpression or small interfering RNA (siRNA)-mediated depletion of ASF/SF2 did not affect the splicing of a WT BRCA1 minigene but resulted in an increase and decrease of T6 exon 18 inclusion, respectively. Furthermore, extensive mutation analysis using hybrid minigenes indicated that the T6 mutant creates a sequence with a prevalently inhibitory function. Indeed, RNA-protein interaction and siRNA experiments showed that the skipping of T6 BRCA1 exon 18 is due to the creation of a splicing factor-dependent silencer. This sequence specifically binds to the known repressor protein hnRNPA1/A2 and to DAZAP1, the involvement of which in splicing inhibition we have demonstrated. Our results indicate that the binding of the splicing factors hnRNPA1/A2 and DAZAP1 is the primary determinant of T6 BRCA1 exon 18 exclusion.

A meta-analysis was performed in order to test the hypothesis that Japanese have a greater amount of abdominal visceral fat (AVF) relative to abdominal subcutaneous fat (ASF) than Caucasians. Data were derived from published studies that included mean values for AVF and ASF areas, measured using computed tomography, and age for native Japanese, African-Americans, and Caucasians of both genders. Mean values from each study were used as single data points. A significant difference in AVF was observed between Japanese and Caucasian populations after adjusting for ASF, age, and sex ( p<0.05). However, the difference in AVF between Japanese and Caucasian females was lower than that between African-American and Caucasian females.

African swine fever (ASF) is a major threat to the pig industry in Europe. Since 2007, ASF outbreaks have been ongoing in the Caucasus, Eastern Europe and the Baltic countries, causing severe economic losses for many pig farmers and pork producers. In addition, the number of ASF cases in wild boar populations has dramatically increased over the past few years. Evidence supports direct contact with infectious domestic pigs and wild boars, and consumption of contaminated feed, as the main transmission routes of ASF virus (ASFV) to domestic pigs. However, significant knowledge gaps highlight the urgent need for research to investigate the dynamics of indirect transmission via the environment, the minimal infective doses for contaminated feed ingestion, the probability of effective contacts between infectious wild boars and domestic pigs, the potential for recovered animals to become carriers and a reservoir for transmission, the potential virus persistence within wild boar populations and the influence of human behaviour for the spread of ASFV. This will provide an improved scientific basis to optimise current interventions and develop new tools and strategies to reduce the risk of ASFV transmission to domestic pigs.

African swine fever virus ASFV/NH/P68 is a naturally occurring, non-haemadsorbing and non-fatal isolate. Longitudinal clinical and immunological studies on 31 pigs inoculated oronasally or intramuscularly with this isolate defined two discrete groups of animals: those developing ASF chronic type lesions and those remaining asymptomatic. Animals developing lesions had viraemia and fever late after infection, NK activity levels close to that of control animals and high levels of anti-ASFV specific antibodies together with a marked hypergammaglobulinaemia involving IgG1, IgG2, IgM and IgA immunoglobulin isotypes. Pigs remaining asymptomatic after infection, on the other hand, did not have viraemia or fever after day 14 post-infection and had elevated NK cell activity, but normal plasma Ig concentrations and relatively low specific anti-virus antibody concentrations throughout the duration of the experiments. Importantly, the latter group of pigs virus were resistant to subsequent challenge with the highly virulent ASFV/L60 isolate and survived with no major changes in any of the parameters examined and referred to above. Finally, lymphoproliferative responses to the mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen were not depressed in either of the two clinically defined groups of pigs. Thus further studies with this infection model may provide new insights on mechanisms of protective immunity to ASFV.

One X chromosome, selected at random, is silenced in each female mammalian cell. Xist encodes a noncoding RNA that influences the probability that the cis-linked X chromosome will be silenced. We found that the A-repeat, a highly conserved element within Xist, is required for the accumulation of spliced Xist RNA. In addition, the A-repeat is necessary for X-inactivation to occur randomly. In combination, our data suggest that normal Xist RNA processing is important in the regulation of random X-inactivation. We propose that modulation of Xist RNA processing may be part of the stochastic process that determines which X chromosome will be inactivated.

In higher plants, activating sequence-1 (as-1) of the cauliflower mosaic virus 35 S promoter mediates both salicylic acid (SA)- and auxin-inducible transcriptional activation. Originally found in promoters of several viral and bacterial plant pathogens, as-1-like elements are also functional elements of plant promoters activated in the course of a defense response upon pathogen attack. Nuclear as-1-binding factor (ASF-1) and cellular salicylic acid response protein (SARP) bind specifically to as-1. Four different tobacco bZIP transcription factors (TGA1a, PG13, TGA2.1, and TGA2.2) are potential components of either ASF-1 or SARP. Here we show that ASF-1 and SARP are very similar in their composition. TGA2.2 is a major component of either complex, as shown by supershift analysis and Western blot analysis of DNA affinity-purified SARP. Minor amounts of a protein immunologically related to TGA2.1 were detected, whereas TGA1a was not detectable. Overexpression of either TGA2.2 or a dominant negative TGA2.2 mutant affected both SA and auxin (2, 4D) inducibility of various target promoters encoding as-1-like elements, albeit to different extents. This indicates that TGA2.2 is a component of the enhancosome assembling on these target promoters, both under elevated SA and 2,4D concentrations. However, the effect of altered TGA2.2 levels on gene expression was more pronounced upon SA treatment than upon 2,4D treatment.

Interaction between transcription and pre-mRNA processing via binding of polymerase II (Pol II) to factors involved in capping, splicing, and polyadenylation has recently been demonstrated. The C-terminal domain (CTD), a highly phosphorylated repeat sequence of the largest subunit of Pol II, has been implicated in this interaction because deletion of this domain affects downstream RNA processing events and because it is the binding site for numerous processing factors. Here we show that recombinant CTD, free of other components of Pol II, activated in vitro splicing and assembly of the spliceosome in nuclear extracts if, and only if, the assayed precursor RNA was recognized via exon definition, i.e., if the substrates contained complete exons with both 3' and 5' splice sites. Furthermore, depletion of intact Pol II inactivated splicing of this set of precursor RNAs and addition of recombinant CTD restored activity. The added recombinant CTD was quickly hyper- and hypophosphorylated in extract, became associated with the precursor RNA, and stimulated the association of U1 snRNPs but not ASF/SF2 with substrate RNA. These observations suggest that the mode of interaction between the CTD and splicing factors is integrally tied to exon definition and the mechanism whereby distal exons can be recognized and brought into juxtaposition during assembly of the spliceosome.

Serine/arginine-rich (SR) proteins are associated with either the regulation or the execution of both constitutive splicing and the selection of alternative splice sites in animals and plants. We demonstrated the molecular characterization of a homolog of SR protein, atSR45a, in Arabidopsis plants. Six types of mRNA variants (atSR45a-1a-e and atSR45a-2) were generated by the alternative selection of transcriptional initiation sites and the alternative splicing of introns in atSR45a pre-mRNA. The atSR45a-1a and -2 proteins, presumed mature forms, were located in the nucleus and interacted with U1-70K, suggesting that these proteins function as a splicing factor in Arabidopsis. The levels of the transcripts atSR45a and atSR30, SF2/ASF-like SR proteins, were increased by various types of stress, such as high-light irradiation and salinity. Furthermore, the splicing patterns of atSR45a and atSR30 pre-mRNA themselves were altered under these stressful conditions. In particular, the expression of atSR45a-1a, atSR45a-2, atSR30 mRNA1 and atSR30 mRNA3 was greatly increased by high-light irradiation. These results indicate that the regulation of transcription and alternative splicing of atSR45a and atSR30 is responsive to various stressful conditions.

SR proteins purified from uninfected HeLa cells inhibit adenovirus IIIa pre-mRNA splicing by binding to the intronic IIIa repressor element (3RE). In contrast, SR proteins purified from late adenovirus-infected cells are functionally inactivated as splicing repressor proteins by a virus-induced dephosphorylation. We have shown that the adenovirus E4-ORF4 protein, which binds the cellular protein phos phatase 2A (PP2A) and activates IIIa splicing in vitro and in vivo, induces SR protein dephosphorylation. Here we show that E4-ORF4 interacts with only a subset of SR proteins present in HeLa cells. Thus, E4-ORF4 interacts efficiently with SF2/ASF and SRp30c, but not with other SR proteins. Interestingly, E4-ORF4 interacts with SF2/ASF through the latter's RNA recognition motifs. Furthermore, E4-ORF4 interacts preferentially with the hyperphosphorylated form of SR proteins found in uninfected HeLa cells. E4-ORF4 mutant proteins that fail to bind strongly to PP2A or SF2/ASF do not relieve the repressive effect of HeLa SR proteins on IIIa pre-mRNA splicing in transient transfection experiments, suggesting that an interaction between all three proteins is required for E4-ORF4-induced SR protein dephosphorylation.

Retroviral replication requires that a portion of the primary transcripts generated from proviral DNA be spliced to serve as mRNA for the envelope protein and in Rous sarcoma virus as src mRNA. However, a substantial amount of full-length RNA must be maintained in an unspliced form, as the unspliced RNA serves both as mRNA for structural proteins and virion-associated enzymatic proteins and as genomic RNA for progeny virions. The extent of viral RNA splicing must be finely controlled, since only a narrow range in the ratio of unspliced RNA to spliced RNA is tolerated for optimal replication. A number of cis-acting sequences within the RNA of Rous sarcoma virus play a role in preserving a large pool of unspliced RNA. One such sequence, the negative regulator of splicing (NRS), is of interest because it blocks splicing but is not located near any of the splice junctions. To better understand how this novel element blocks splicing at a distance, we set out to identify host cell factors that interact specifically with this inhibitory sequence. In this study, proteins from nuclear extracts with molecular masses of 26, 36, 44, and 55 kDa were shown by UV cross-linking assays to bind the NRS preferentially. One of them, p55, was also detected in a specific complex with SR protein electrophoretic mobility shift assay. All but p55 have biochemical properties consistent with SR protein splicing factors, and some, but not all, of the total SR proteins purified from HeLa cells cross-link specifically to the NRS. The strongest cross-linking SR protein is SRp30a/b, which is composed of the splicing factors SF2/ASF and SC35. The NRS specifically binds bacterially expressed SF2/ASF, whereas nonfunctional mutants do not. Data indicating that the 36-kDa protein which cross-links in nuclear extracts is SF2/ASF are presented. The data indicate that factors normally required for RNA splicing may be exploited by retroviruses to block splicing.

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

The small GTPase Rac1 regulates signaling pathways controlling actin-dependent cell motility as well as gene transcription. An alternative splicing variant Rac1b is overexpressed in a subset of colorectal tumors and is required to sustain tumor cell viability. Thus, it is of therapeutic interest to understand the molecular mechanism behind the overexpression of Rac1b through alternative splicing. Here we describe that ASF/SF2 and SRp20 are two antagonistic splicing factors regulating Rac1b expression in colorectal tumor cells. Using an Rac1 minigene, we identified that SRp20 increased skipping of alternative exon 3b in HT29 colorectal cells, whereas ASF/SF2 increased its inclusion. The depletion of the endogenous expression of these splicing factors by specific small interfering RNA confirmed that ASF/SF2 acts as an enhancer of endogenous Rac1b splicing, whereas SRp20 acts as a silencer. Point mutations in exon 3b defined two adjacent regulatory regions required for skipping or inclusion of exon 3b, which are recognized in vitro by SRp20 and ASF/SF2, respectively. Both splicing factors were found to be regulated by upstream signaling pathways: the inhibition of the phosphatidylinositol 3-kinase pathway increased protein levels of ASF/SF2 and promoted Rac1b, whereas activation of beta-catenin/TCF4 increased expression of SRp20 and inhibited that of Rac1b. Together, these data reveal that signaling pathways act in concert to target independent splicing factors and achieve the correct combinatorial code to regulate alternative splicing of the small GTPase Rac1.