African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal viral disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Successful experimental vaccines have been derived from naturally occurring, cell culture-adapted, or genetically modified live attenuated ASFV. Recombinant viruses harboring engineered deletions of specific virulence-associated genes induce solid protection against challenge with parental viruses. Deletion of the 9GL (B119L) gene in the highly virulent ASFV isolates Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Δ9GL viruses) resulted in complete protection when challenged with parental isolates. When similar deletions were created within the ASFV Georgia 2007 (ASFV-G) genome, attenuation was achieved but the protective and lethal doses were too similar. To enhance attenuation of ASFV-G, we deleted another gene, UK (DP96R), which was previously shown to be involved in attenuation of the ASFV E70 isolate. Here, we report the construction of a double-gene-deletion recombinant virus, ASFV-G-Δ9GL/ΔUK. When administered intramuscularly (i.m.) to swine, there was no induction of disease, even at high doses (106 HAD50). Importantly, animals infected with 104 50% hemadsorbing doses (HAD50) of ASFV-G-Δ9GL/ΔUK were protected as early as 14 days postinoculation when challenged with ASFV-G. The presence of protection correlates with the appearance of serum anti-ASFV antibodies, but not with virus-specific circulating ASFV-specific gamma interferon (IFN-γ)-producing cells. ASFV-G-Δ9GL/ΔUK is the first rationally designed experimental ASFV vaccine that protects against the highly virulent ASFV Georgia 2007 isolate as early as 2 weeks postvaccination.

OBJECTIVE

Currently, there is no commercially available vaccine against African swine fever. Outbreaks of the disease are devastating to the swine industry and are caused by circulating strains of African swine fever virus. Here, we report a putative vaccine derived from a currently circulating strain but containing two deletions in two separate areas of the virus, allowing increased safety. Using this genetically modified virus, we were able to vaccinate swine and protect them from developing ASF. We were able to achieve protection from disease as early as 2 weeks after vaccination, even when the pigs were exposed to a higher than normal concentration of ASFV.

The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.

Wild primates take most of the daily diet from plant sources, eating moderate to small amounts of animal source foods (ASF). Plant materials make up from 87% to >99% of the annual diet of great apes, the closest living relatives of modern humans (Homo sapiens sapiens). Reflecting their close genetic relationship, gut form and nutrient requirements of apes and humans (Hominoidea) are very similar, as is their pattern of digestive kinetics-one predicated on a relatively slow turnover of ingesta. In plant-eating mammals, in contrast to carnivorous mammals, greater body size is associated with lower dietary quality. Turning to ASF as a routine rather than occasional dietary component would have permitted the evolving human lineage to evade the nutritional constraints placed on body size increases in apes. Without routine access to ASF, it is highly unlikely that evolving humans could have achieved their unusually large and complex brain while simultaneously continuing their evolutionary trajectory as large, active and highly social primates. As human evolution progressed, young children in particular, with their rapidly expanding large brain and high metabolic and nutritional demands relative to adults would have benefited from volumetrically concentrated, high quality foods such as meat. Today, many humans, particularly those in high income nations, have a variety of high quality, non-ASF dietary alternatives, but such foods were not generally available to paleolithic human ancestors nor to many people today in low income nations.

Missense, nonsense, and translationally silent mutations can inactivate genes by altering the inclusion of mutant exons in mRNA, but their overall frequency among disease-causing exonic substitutions is unknown. Here, we have tested missense and silent mutations deposited in the BRCA1 mutation databases of unclassified variants for their effects on exon inclusion. Analysis of 21 BRCA1 variants using minigene assays revealed a single exon-skipping mutation c.231G>T. Comprehensive mutagenesis of an adjacent 12-nt segment showed that this silent mutation resulted in a higher level of exon skipping than the 35 other single-nucleotide substitutions. Exon inclusion levels of mutant constructs correlated significantly with predicted splicing enhancers/silencers, prompting the development of two online utilities freely available at http://www.dbass.org.uk. EX-SKIP quickly estimates which allele is more susceptible to exon skipping, whereas HOT-SKIP examines all possible mutations at each exon position and identifies candidate exon-skipping positions/substitutions. We demonstrate that the distribution of exon-skipping and disease-associated substitutions previously identified in coding regions was biased toward top-ranking HOT-SKIP mutations. Finally, we show that proteins 9G8, SC35, SF2/ASF, Tra2, and hnRNP A1 were associated with significant alterations of BRCA1 exon 6 inclusion in the mRNA. Together, these results facilitate prediction of exonic substitutions that reduce exon inclusion in mature transcripts.

An experimental infection was conducted to evaluate horizontal transmission, clinical, virological and humoral response induced in domestic pigs infected with African swine fever (ASF) genotype II virus circulating in 2014 into the European Union (EU). Ten naive pigs were placed in contact with eight pigs experimentally inoculated with the Lithuanian LT14/1490 ASF virus (ASFV) responsible for the first ASF case detected in wild boar in Lithuania in January 2014. Clinical examination and rectal temperature were recorded each day. Blood sampling from every animal was carried out twice weekly. Blood samples were examined for presence of ASF virus-specific antibodies and for determining the ASFV viral load. From the obtained results, it was concluded that the Lithuanian ASFV induced an acute disease which resulted in 94, 5% mortality. The disease was easily detected by real-time PCR prior to the onset of clinical signs and 33% of the animals seroconverted. All findings were in accordance with observations previously made in domestic pigs and wild boar when infected with ASF genotype II viruses characterized by a high virulence. One in-contact pig remained asymptomatic and survived the infection. The role of such animals in virus transmission would need further investigation.

There has been an evolution in our understanding of the causes of undernutrition and of the nutrition intervention programs that should be prioritized. This article discusses why nutrition programs have shifted their primary emphasis from control of protein deficiency, to energy deficiency, and now to micronutrient deficiencies. It has become recognized by the nutrition community that micronutrient malnutrition is very widespread, and is probably the main nutritional problem in the world. The most commonly used strategies for micronutrient deficiency control are supplementation and fortification, because they are cost-effective and to some extent, relatively easy to deliver. They have important limitations, however. Relatively little emphasis has been placed on food-based approaches to control micronutrient malnutrition. Evidence from several past studies, including the Nutrition Collaborative Research Support Program (N-CRSP), indicated a strong positive association between animal source food (ASF) intake, micronutrient status, and many human functions. This association motivated the intervention studies supported by the Global Livestock CRSP and described in this supplement, which found benefits of increasing ASF intake. In contrast to the common assumption that increasing consumption of ASF in poor communities is infeasible, and will only cause environmental problems, the articles in this supplement show the potential economic benefits of animal ownership in poor communities, and provide examples of innovative programs that have increased local production and consumption. Much more communication is needed among the nutrition, agriculture and development communities to achieve improved dietary quality for poor populations.

Alterations in alternative splicing affect essential biologic processes and are the basis for a number of pathologic conditions, including cancer. In this review we will summarize the evidence supporting the relevance of alternative splicing in lung cancer. An example that illustrates this relevance is the altered balance between Bcl-xL and Bcl-xS, two splice variants of the apoptosis regulator Bcl-x. Splice modifications in cancer-related genes can be associated with modifications either in cis-acting splicing regulatory sequences or in trans-acting splicing factors. In fact, lung tumors show abnormal expression of splicing regulators such as ASF/SF2 or some members of the heterogeneous nuclear ribonucleoprotein family. The potential significance of alternative splicing as a target for lung cancer diagnosis or treatment will also be discussed.

Two-dimensional (2D) analysis of African swine fever (ASF) virus purified by Percoll gradient centrifugation resolves 54 structural proteins, 30 in conventional IEF gels and 24 in NEPHGE gels, while only 26 structural proteins are separated by SDS-PAGE. The two main bands separated by SDS-PAGE, with mol wt 150K and 72K, correspond to single spots in 2D gels. Other bands, including major bands of 38K, 35K, 24K, 17K, and 15.5K mol wt, correspond to multiple proteins of the same molecular weight but different pI. One hundred six virus-specific proteins were resolved by 2D analysis, 59 in conventional IEF gels and 47 in NEPHGE gels. Thirty-five of the virus-specific proteins are early proteins, synthesized before DNA replication, and the remaining 71 proteins are late proteins. Early proteins belong to two groups: 11 transient early proteins are synthesized only early in infection and the other 24 are persistent early proteins, synthesized at both early and late phases. Treatment with cytosine arabinoside prevents the synthesis of late proteins and blocks the shut-off of the synthesis of transient early proteins. Eleven structural proteins are major early proteins and 28 are late proteins. The remaining 15 structural proteins migrate in 2D gels like cellular proteins. Three of these cellular proteins, with mol wt 58K, 56K, and 45K were identified by immunoblotting as alpha-tubulin, beta-tubulin, and actin, respectively.

The human alternative splicing factor ASF/SF2, an SR (serine-arginine-rich) protein involved in mRNA splicing control, is activated by the multisite phosphorylation of its C-terminal RS domain, a segment containing numerous arginine-serine dipeptide repeats. The protein kinase responsible for this modification, SR-specific protein kinase 1 (SRPK1), catalyzes the selective phosphorylation of approximately a dozen serines in only the N-terminal portion of the RS domain (RS1). To gain insights into the nature of selective phosphate incorporation in ASF/SF2, region-specific phosphorylation in the RS domain was monitored as a function of reaction progress. Arg-to-Lys mutations were made at several positions to produce unique protease cleavage sites that separate the RS domain into identifiable N- and C-terminal phosphopeptides upon treatment with lysyl endoproteinase. These studies reveal that SRPK1 docks near the C-terminus of the RS1 segment and then moves in an N-terminal direction along the RS domain. Multiple quadruple Ser-to-Ala and deletion mutations did not disrupt the phosphorylation of other sites regardless of position, suggesting that the active site of SRPK1 docks in a flexible manner at the center of the RS domain. Taken together, these data suggest that SRPK1 uses a unique 'grab-and-pull' mechanism to control the regiospecific phosphorylation of its protein substrate.

Alternative splicing is an essential process that produces protein diversity in humans. It is also the cause of many complex diseases. Spinal muscular atrophy (SMA), the second most common autosomal recessive disorder, is caused by the absence of or mutations in the Survival Motor Neuron 1 (SMN1) gene, which encodes an essential protein. A nearly identical copy of the gene, SMN2, fails to compensate for the loss of SMN1 because exon 7 is alternatively spliced, producing a truncated protein, which is unstable. SMN1 and SMN2 differ by a critical C-to-T substitution at position 6 of exon 7 in SMN2 (C6U transition in mRNA). This substitution alone is enough to cause an exon 7 exclusion in SMN2. Various cis- and trans-acting factors have been shown to neutralize the inhibitory effects of C6U transition. Published reports propose models in which either abrogation of an enhancer element associated with SF2/ASF or gain of a silencer element associated with hnRNP A1 is the major cause of exon 7 exclusion in SMN2. Most recent model suggests the presence of an EXtended INhibitory ContexT (Exinct) that is formed as a consequence of C6U transition in exon 7 of SMN2. In Exinct model, several factors may affect exon 7 splicing through cooperative interactions. Such regulation may be common to many alternatively spliced exons in humans. Recent advances in our understanding of SMN gene splicing reveals multiple challenges that are specific to in vivo regulation, which we now know is intimately connected with other biological pathways.

Alternative splicing is part of the expression program of a wide number of genes implicated in cell growth and differentiation. Although the occurrence of inappropriate alternative splicing in tumors has started to emerge, the underlying molecular mechanisms have been, thus far, largely unexplored. We have investigated the alternative splicing pattern of the CD44 gene in specimens of nonfamilial colon adenocarcinomas at different stages of tumor progression. In the same patients, we have assessed by Northern blotting analysis the mRNA levels of different heterogeneous nuclear ribonucleoproteins and SR factors, all involved in pre-mRNA splicing and, more in general, in mRNA maturation. The results of this analysis highlight a general rule for the mode of splicing of the CD44 pre-mRNA. Moreover, we found that the mRNA levels of different SR proteins in tumor specimens are different from, and usually lower than, those detected in samples of nonpathological tissue adjacent to the tumor. Quantitative analysis demonstrates that, in tumors, the mRNA levels of ASF, SRp40, SRp55, and SRp75, when normalized to those of heterogeneous nuclear ribonucleoprotein A1, are lower than those of SRp20 and SRp30. Interestingly, this reduction is more drastic in patients showing a more altered CD44 splicing pattern and seems to be related to the propensity to develop metastases.

Production of the structural and enzymatic proteins of type 1 human immunodeficiency virus (HIV-1) is controlled by the rev regulatory gene product. The 116-amino acid Rev protein acts by binding to the Rev response element (RRE), a complex RNA stem-loop structure located within the env gene of HIV. Rev exerts a series of posttranscriptional effects, including the inhibition of viral RNA splicing, the activation of nuclear export of incompletely spliced viral RNAs, and the enhancement of translation of RRE-containing RNAs. Our studies now demonstrate that at least one member of the SR family of splicing factors, SF2/ASF, specifically binds to a subregion of the RRE in vitro in a Rev-dependent manner. Furthermore, expression of high levels of SF2/ASF inhibits Rev function and impairs HIV replication in vivo. Both the in vitro binding of SF2/ASF to the Rev/RRE complex and the in vivo inhibition of Rev action by SF2/ASF are abrogated by mutation of the N-terminal RNA recognition motif but are not affected by mutation of the C-terminal arginine-serine-rich domain. These findings suggest that Rev inhibition of HIV splicing likely involves recruitment of the essential splicing factor SF2/ASF to the Rev/RRE complex. However, these inhibitory effects of Rev on viral RNA splicing are apparently overcome by augmenting the intracellular levels of SF2/ASF expression.

We have isolated and characterized a tobacco gene, designated G13, encoding a leucine zipper DNA-binding protein related to the transcription activator TGA1a. The G13 coding region is divided into eight exons and the amino acid sequence of the encoded protein (PG13) shows 76% homology to TGA1a. Their putative DNA-contacting regions (basic domains) are identical and they both bind to the same target sequences in vitro. By contrast, some differences are apparent between these proteins at the carboxyl end of the dimerization region (leucine zipper). The basic and leucine zipper domains are encoded on separate small exons. Analysis by DNAse I footprinting, gel shift and competition experiments revealed that TGA1a and PG13 synthesized in Escherichia coli, and the tobacco nuclear factor ASF-1 all bind to at least one site in the 5' upstream region of G13. The presence of a TGA1a binding site in the upstream region of a TGA1a-related gene suggests that transcription of this gene is autoregulated.

The SR (arginine-serine rich) protein ASF/SF2 (also called human alternative splicing factor), an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRMs; RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain, a domain rich in arginine-serine repeats). The SR-specific protein kinase (SRPK) 1 phosphorylates the RS domain at multiple serines using a directional (C-terminal-to-N-terminal) and processive mechanism--a process that directs the SR protein to the nucleus and influences protein-protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme-substrate complex was analyzed using single-turnover and multiturnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1-RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible protein-protein interactions to facilitate both early and late phases of the processive phosphorylation of SR proteins.

Human papillomavirus (HPV) gene expression is regulated in concert with the epithelial differentiation program. In particular, expression of the virus capsid proteins L1 and L2 is tightly restricted to differentiated epithelial cells. For HPV16, the capsid proteins are encoded by 13 structurally different mRNAs that are produced by extensive alternative splicing. Previously, we demonstrated that upon epithelial differentiation, HPV16 infection upregulates hnRNP A1 and SF2/ASF, both key factors in alternative splicing regulation. Here we cloned a 1-kb region upstream of and including the transcriptional start site of the SF2ASF gene and used it in in vivo transcription assays to demonstrate that the HPV16 E2 transcription factor transactivates the SF2/ASF promoter. The transactivation domain but not the DNA binding domain of the protein is necessary for this. Active E2 association with the promoter was demonstrated using chromatin immunoprecipitation assays. Electrophoretic mobility shift assays indicated that E2 interacted with a region 482 to 684 bp upstream of the transcription initiation site in vitro. This is the first time that HPV16 E2 has been shown to regulate cellular gene expression and the first report of viral regulation of expression of an RNA processing factor. Such E2-mediated control during differentiation of infected epithelial cells may facilitate late capsid protein expression and completion of the virus life cycle.

Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

Due to the strong sequence homology it has been suggested that CDC2L5 and CDK12 belong to a high molecular weight subfamily of CDC2 family with PITAI/VRE motifs [F. Marques, J.L. Moreau, G. Peaucellier, J.C. Lozano, P. Schatt, A. Picard, I. Callebaut, E. Perret, A.M. Geneviere, A new subfamily of high molecular mass CDC2-related kinases with PITAI/VRE motifs, Biochem. Biophys. Res. Commun. 279 (2000) 832-837]. Recently, we reported that CDK12 interacts with L-type cyclins and is involved in alternative splicing regulation [H.-H. Chen, Y.-C. Wang, M.-J. Fann, Identification and characterization of the CDK12/Cyclin L1 complex involved in alternative splicing regulation, Mol. Cel. Biol. 26 (2006) 2736-2745]. Here, we provide evidence that CDC2L5 also interacts with L-type cyclins and thus rename it as cyclin-dependent kinase 13 (CDK13). The kinase domain of CDK13 is sufficient to bind the cyclin domains of L-type cyclins. Moreover, CDK13 and L-type cyclins modulate each other's subcellular localization. When CDK13 and an E1a minigene reporter construct were over-expressed in HEK293T cells, CDK13 alters the splicing pattern of E1a transcripts in a dose-dependent manner. Similar to effects of CDK12, effects of CDK13 on splicing pattern are counteracted by SF2/ASF and SC35. These findings strengthen CDK12 and CDK13 as a subfamily of cyclin-dependent kinases that regulate alternative splicing.

Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-beta, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten(-/-) fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten(-/-) cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten(-/-) cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in cross-linking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here we studied interaction of galectin-3 with the model glycoprotein asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concentration of free galectin carbohydrate recognition sites in solution. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concentrations, showing that many more galectin-3 molecules were engaged than expected due to the about nine known glycan-based binding sites per ASF molecule. This suggests that after ASF-induced nucleation, galectin-3 associates with itself by the carbohydrate recognition site binding to another galectin-3 molecule, possibly forming oligomers. We named this type-C self-association to distinguish it from the previously proposed models (type-N) where galectin-3 molecules bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-association can result in precipitates, as measured here by turbidimetry and dynamic light scattering. Type-C self-association and precipitation occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required much higher concentration (∼50 μM) as compared with wild type (∼1 μM). ASF also induced weaker type-C self-association of galectin-3 lacking its N-terminal domains, but as expected, no precipitation. Neither a monovalent nor a divalent N-acetyl-D-lactosamine-containing glycan induced type-C self-association, even if the latter gave precipitates with high concentrations of galectin-3 >>∼50 μM) in agreement with published results and perhaps due to type-N self-association.

Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3' splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2alpha and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.

Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites. They are recognized by members of the serine/arginine-rich (SR) family of proteins, such as splicing factor 2/alternative splicing factor (SF2/ASF), which recruit basal splicing factors to form the initial complexes during spliceosome assembly. The in vitro splicing kinetics of an ESE-dependent IgM pre-mRNA suggested that an SF2/ASF-specific ESE has additional functions later in the splicing reaction, after the completion of the first catalytic step. A bimolecular exon ligation assay, which physically uncouples the first and second catalytic steps of splicing in a trans-splicing reaction, was adapted to test the function of the ESE after the first step. A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not. The ESE-dependent trans-splicing reaction occurred after inactivation of U1 or U2 small nuclear ribonucleoprotein particles, compatible with a functional assay for events after the first step of splicing. The ESE-dependent step appears to take place before the ATP-independent part of the second catalytic step. Bimolecular exon ligation also occurred in an S100 cytosolic extract, requiring both the SF2/ASF-dependent ESE and complementation with SF2/ASF. These data suggest that some ESEs can act late in the splicing reaction, together with appropriate SR proteins, to enhance the second catalytic step of splicing.

Tau exon 10 splicing is altered by autosomal dominant mutations that cause frontotemporal dementia with parkinsonism chromosome 17-type and by unknown mechanisms in other related neurodegenerative disorders. Identifying cis- and trans-regulators of tau exon 10 splicing is therefore crucial for understanding disease mechanisms. We previously identified several splicing enhancers and silencers within exon 10 and intron 10. Here, we show that splicing factors SF2/ASF, Tra2beta, and a 50-kDa nuclear protein bind in vitro to the polypurine enhancer at the 5' end of exon 10. Disease splicing mutations N279K and Delta280K disrupt the enhancer and alter associations with these factors. N279K targets robustly bind Tra2beta compared with the normal enhancer, which may explain why N279K enhances exon 10 splicing in vivo. In contrast, factor associations with Delta280K targets are nearly undetectable, explaining why Delta280K almost abolishes exon 10 splicing in vivo. Small interfering RNA-mediated suppression of endogenous SF2/ASF and Tra2beta significantly reduces exon 10 splicing. Exogenous SF2/ASF dramatically enhances normal exon 10 splicing and efficiently rescues the Delta280K splicing defect. Domain deletion analyses show that the C-terminal RS domains of SF2/ASF and Tra2beta are required for normal exon 10 splicing in vivo. In contrast to Tra2beta, the SF2/ASF RS domain remains essential in the presence of a strengthened enhancer or when either weak splice site is strengthened. The data suggest that SF2/ASF has both essential and regulatory roles, whereas Tra2beta has a supporting role in exon 10 splicing.

The large subunit rRNA in eukaryotes contains an unusually dense cluster of 8-10 pseudouridine (Psi) modifications located in a three-helix structure (H37-H39) implicated in several functions. This region is dominated by a long flexible helix (H38) known as the "A-site finger" (ASF). The ASF protrudes from the large subunit just above the A-site of tRNA binding, interacts with 5 S rRNA and tRNA, and through the terminal loop, forms a bridge (B1a) with the small subunit. In yeast, the three-helix domain contains 10 Psis and 6 are concentrated in the ASF helix (3 of the ASF Psis are conserved among eukaryotes). Here, we show by genetic depletion analysis that the Psis in the ASF helix and adjoining helices are not crucial for cell viability; however, their presence notably enhances ribosome fitness. Depleting different combinations of Psis suggest that the modification pattern is important and revealed that loss of multiple Psis negatively influences ribosome performance. The effects observed include slower cell growth (reduced rates up to 23% at 30 degrees C and 40-50% at 37 degrees C and 11 degrees C), reduced level of the large subunit (up to 17%), impaired polysome formation (appearance of half-mers), reduced translation activity (up to 20% at 30 degrees C and 25% at 11 degrees C), and increased sensitivity to ribosome-based drugs. The results indicate that the Psis in the three-helix region improve fitness of a eukaryotic ribosome.

The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3' region resembles a minor class 5' splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5' half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3' region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF.