Vascular endothelial growth factor-A (VEGF-A) has several isoforms, which differ in their capacity to bind extracellular matrix proteins and also in their affinity for VEGF receptors. Although the relative contribution of the VEGF isoforms has been studied in tumor angiogenesis, little is known about the mechanisms that regulate the alternative splicing process. Here, we tested microenvironment cues that might regulate VEGF alternative splicing. To test this, we used endometrial cancer cells that produce all VEGF isoforms as a model, and exposed them to varying pH levels, hormones, glucose and CoCl(2) (to mimic hypoxia). Low pH had the most consistent effects in inducing variations in VEGF splicing pattern (VEGF121 increased significantly, p < 0.001, when compared to VEGF145, 165 or 189). This was accompanied by activation of the p38 stress pathway and SR proteins (splicing factors) expression and phosphorylation. SF2/ASF, SRp20 and SRp40 down-regulation by siRNA impaired the effects of pH stimulation, blocking the shift in VEGF isoforms production. Taken together, we show for the first time that acidosis (low pH) regulates VEGF-A alternative splicing, may be through p38 activation and suggest the possible SR proteins involved in this process.

African swine fever is viral disease of domestic and wild pigs which leads to almost total mortality and causes great economic losses due to absence of vaccine. Having been introduced into the Russian Federation in 2007 the disease has spread widely in the southern region of the country and since 2011 has demonstrated a tendency to form a secondary endemic zone in the central part of the country. In the present study spatio-temporal patterns of ASF diffusion in the populations of wild and domestic pigs are analyzed. The structure of the domestic swine population is conventionally divided into a sub-population at low biosecurity (77% of the total number of outbreaks in domestic pigs) and a population at high biosecurity (23%). The statistics of ASF cases registered in each of these sub-populations is presented. The possible causes of ASF diffusion across the country are discussed. The use of geo-information technologies (GIS) enabled confirmation of the conclusion that an epidemic center has shifted into the central part of Russia. The main conclusions of this study are that: (1) anthropogenic factors play the leading role in the spread of ASF across the territory of the RF; (2) small-scale private holdings (low biosecurity population) are more exposed to ASF virus introduction; (3) there is a high risk of diffusion of ASFV from the secondary endemic zone in the central part of the RF to neighboring regions.

The tumor suppressor protein p53 plays a central role in the cellular response to genotoxic lesions and has been shown to be activated by most anticancer agents such as mitomycin C. We here show that mitomycin C treatment of human MCF7 breast adenocarcinoma cells results in increased topoisomerase I activity as measured by relaxation of supercoiled DNA and by phosphorylation of SR protein splicing factor. The increase in catalytic activity occurs in parallel with the nuclear accumulation of p53, resulting in detectable activation of topoisomerase I within less than 1 h of drug treatment. Furthermore, topoisomerase I co-immunoprecipitates with nuclear p53, suggesting that the activation of topoisomerase I may be a result of a direct interaction between the two proteins. In vitro experiments with purified recombinant proteins show that p53 increases the catalytic activities of topoisomerase I as measured by relaxation of supercoiled DNA, stabilization of the covalent topoisomerase I-DNA complex (in the presence of camptothecin), and phosphorylation of SR protein splicing factor ASF/SF2. Furthermore, topoisomerase I sediments at a higher molecular weight in the presence of p53 as revealed by sucrose density gradient analysis in the absence of DNA. Finally, p53 modifies the thermal stability of topoisomerase I, protecting it from heat denaturation. Taken together, our results show that topoisomerase I and p53 form molecular complexes in vitro as in vivo, and we suggest that the p53-mediated response to DNA damage may, at least in part, involve activation of topoisomerase I.

African swine fever (ASF) virus was grown either in swine macrophages or in VERO cells and purified free of cell DNA. Virus DNA was isolated from virions as a molecule with a sedimentation coefficient of 60S and a contour of 58 +/- 3 mum. .these two values give a mol. wt. of 102 +/- 5 X 10(6) and 107 +/- 5 X 10(6), respectively, for the genome of ASF virus. Denatured DNA fragments from ASF virus reassociate with a C0t1/2 value of 0-60 +/- 0-05 MS, which compared with the corresponding value for T4 DNA gives for the molecular mass of ASF virus DNA a value of 102 +/- 8 X 10(6) daltons. Only virus DNA is synthesized ASF virus-infected swine macrophages.

The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several cis elements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterologous introns in vivo and in vitro. Previous data showed that the splicing factors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 binding and activity, suggesting that these factors are important for function. These observations, and the finding that a large spliceosome-like complex (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5' splice site. One model to explain NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3' splice sites. In this study, we provide evidence that the NRS interacts with an adenovirus 3' splice site. The interaction was dependent on the integrity of the branch point and pyrimidine tract of the 3' splice site, and it was sensitive to a mutation that was previously shown to abolish U11 snRNP binding and NRS function. However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3' splice site correlated with U1, not U11, binding. These results show that the NRS can interact with a 3' splice site and suggest that U1 is of primary importance for NRS splicing inhibition.

African swine fever (ASF) was first detected in the Estonian wild boar population in September 2014, while the first domestic pig farm was affected in July 2015. In the present study, we aimed to analyse, retrospectively, the epidemiology of the disease in all 26 outbreaks in domestic pig herds that occurred in Estonia during the period 2015-2017. Formal interviews were conducted to estimate the high-risk period for every farm, and to identify the possible origin of the ASF virus and the mode of virus introduction. Furthermore, the clinical manifestation of the disease as well as the course of the disease within the farm were investigated. Survival analysis was used to calculate herd incidence and to estimate outbreak risk. A hierarchical Bayesian space-time model was used to analyse the associations between outbreaks and ASF occurrence in wild boar. The spatial and temporal distribution of outbreaks was analysed to characterise the ASF epidemic in the Estonian domestic pig population from 2015 to 2017. The estimated high-risk period varied from seven to 20 days with a median of 11 days. On most of the affected farms, the first clinical signs were mild and not specific to ASF despite the high virulence of the circulating virus. Morbidity and mortality were often limited to a single pen or unit of the farm. The highest mortality (29.7%) was seen on backyard farms with 1-10 pigs and the lowest (0.7%) on large commercial farms (>1000 pigs). The spread of the virus within affected farms has been slow and the contagiousness of the virus has been relatively low. Farms of all sizes and types have been at risk, including large commercial farms operating at a high biosecurity level. In none of the affected farms could the specific route of introduction be verified. However, the findings suggested that virus introduction occurred via indirect transmission routes due to insufficient biosecurity. The total herd incidence of outbreaks was similar across all three years, being 2.4% in 2015 and 2016, and 2.0% in 2017. All outbreaks occurred from June to September, during the warmest period of the year. The results suggest that the increase in ASF cases in local wild boar populations is the main risk factor leading to the infection of farms; 88% of outbreaks occurred in areas where ASF virus was detected in wild boar prior to the outbreak, within a radius of 15 km from the outbreak farm.

Despite the implementation of control efforts and funds to fight against the disease, African swine fever (ASF) has been present in Sardinia since 1978. It has caused serious problems for both the industrial pig sector and the regional authorities in Sardinia, as well as the economy of Italy and the European Union, which annually supports the costly eradication programme. During this time, ASF has persisted, especially in the central-east part of Sardinia where almost 75% of the total outbreaks are concentrated. The Sardinian pig sector is clearly divided into two categories based on the specialization and industrialization of production: industrial farms, which represents only 1.8% of the farms in the island and non-professional holdings, which are comprised of small producers (90% of pig holdings have <15 pigs) and apply little to no biosecurity measures. Additionally, illegally raised pigs are still bred in free-ranging systems in certain isolated parts of the island, despite strict regulations. The illegal raising of pigs, along with other high-risk management practices (e.g., use of communal areas) are likely the primary reasons for endemic persistence of the virus in this area. The compensation provided to the farmers, and other aspects of the eradication programme have also negatively influenced eradication efforts, indicating that socio-cultural and economic factors play an important role in the epidemiology of ASF on the island. The aim of this study was to comprehensively review the evolution of the 35-year presence of ASF in Sardinia, including control measures, and the environmental and socio-economic factors that may have contributed to disease endemicity on the island. The present review highlights the need for a coordinated programme that considers these socio-economic and environmental factors and includes an assessment of new cost-effective control strategies and diagnostic tools for effectively controlling ASF in Sardinia.

Cancer cachexia is a multifactorial syndrome that includes muscle wasting and inflammation. As gut microbes influence host immunity and metabolism, we investigated the role of the gut microbiota in the therapeutic management of cancer and associated cachexia. A community-wide analysis of the caecal microbiome in two mouse models of cancer cachexia (acute leukaemia or subcutaneous transplantation of colon cancer cells) identified common microbial signatures, including decreased Lactobacillus spp. and increased Enterobacteriaceae and Parabacteroides goldsteinii/ASF 519. Building on this information, we administered a synbiotic containing inulin-type fructans and live Lactobacillus reuteri 100-23 to leukaemic mice. This treatment restored the Lactobacillus population and reduced the Enterobacteriaceae levels. It also reduced hepatic cancer cell proliferation, muscle wasting and morbidity, and prolonged survival. Administration of the synbiotic was associated with restoration of the expression of antimicrobial proteins controlling intestinal barrier function and gut immunity markers, but did not impact the portal metabolomics imprinting of energy demand. In summary, this study provided evidence that the development of cancer outside the gut can impact intestinal homeostasis and the gut microbial ecosystem and that a synbiotic intervention, by targeting some alterations of the gut microbiota, confers benefits to the host, prolonging survival and reducing cancer proliferation and cachexia.

Two of the 22 presently recognised African swine fever (ASF) virus p72 genotypes are genetically homogeneous and are associated with domestic pig cycles. Of these, genotype VIII comprises just two p72 variants, designated 'a' and 'b' in this study, and is confined to four East African countries where it has caused numerous outbreaks between 1961 and 2001. In order to resolve relationships within this homogeneous genotype, the central variable region (CVR) of the 9RL open reading frame of 38 viruses was characterised and the resulting dataset complemented with seven published sequences. Phylogenetic analysis of the 45 taxa resulted in seven discrete amino acid CVR lineages (A-G). CVR lineage F, 84 amino acids in length and spanning a 40-year period, comprised 26 isolates from Malawi, Mozambique, Zambia and Zimbabwe. The second largest lineage (E), consisted of 10 viruses causing outbreaks over a 10-year period in Zambia, Malawi and Mozambique whilst the remaining five lineages were country-specific and represented by four or less viruses with a maximum circulation period of three years. A combined p72-CVR analysis resulted in eight discrete lineages corresponding to eight unique p72-CVR combinations. One of these, b-F, appears to have arisen by convergent evolution or through an intra-genotypic recombination event, as the individual p72 and CVR gene phylogenies are incongruent. This raises the possibility of intra-genotypic recombination in ASF viruses for the first time. However, given the repetitive nature of the CVR region, convergent evolution cannot be excluded and may be the more likely explanation.

African Swine Fever (ASF) is a highly contagious disease that affects the domestic pig and wild boar population. The aim of this study was to describe the introduction and spread of the ASF virus in Western Europe (1960⁻1995) and in Eastern Europe (2007⁻2018), with particular emphasis on the current ASF situation in Poland and its challenges and future perspectives. The first ASF outbreak in Europe was reported in Portugal in 1957, with the virus spreading over most of Western Europe over the next 30 years. In Eastern Europe, the virus was first observed in Georgia in 2007, from where the disease spread quickly to other neighboring countries, reaching Poland in 2014. Since then, there have been 3341 confirmed cases in the wild boar population in Poland. Although there have been no confirmed cases of wild boars coming into contact with domestic pigs, the first notified case concerning domestic pigs was reported in July 2014. Since then, there have been a total of 213 confirmed outbreaks of ASF on Polish pig farms. Given the virulence of the ASF virus and the myriad of transmission routes across Europe, the monitoring of this disease must be a priority for Europe.

Heterogeneous nuclear ribonucleoparticule A1/A2 (hnRNP A1/A2) and splicing factor 2/alternative splicing factor (SF2/ASF) are pivotal for precursor messenger RNA (pre-mRNA) splicing. Interferon regulatory factor-3 (IRF-3) plays critical roles in host defense against viral and microbial infection. Truncated IRF-3 proteins resulting from alternative splicing have been identified and characterized as functional antagonists to full-length IRF-3. In this study, we examined the molecular mechanism for splicing regulation of IRF-3 pre-mRNA and first reported the regulatory effect of hnRNP A1/A2 and SF2/ASF on IRF-3 splicing and activation. RNA interference-mediated depletion of hnRNP A1/A2 or SF2/ASF in human non-small cell lung cancer (NSCLC) cells increased exclusion of exons 2 and 3 of IRF-3 gene and reduced expression levels of IRF-3 protein and IRF-3 downstream effector molecules interferon-beta and CXCL10/IP-10. In addition, direct binding of hnRNP A1 and SF2/ASF to specific binding motifs in IRF-3 intron 1 was confirmed by RNA electrophoretic mobility shift assay. Subsequent minigene splicing assay showed that IRF-3 minigenes with mutated hnRNPA 1/A2 or SF2/ASF binding motifs increased exclusion of exons 2 and 3. Moreover, knockdown of hnRNP A1/A2 or SF2/ASF in NSCLC cells reinforced phytohemagglutinin-induced tumor necrosis factor-alpha release by peripheral blood mononuclear cells (PBMC) but suppressed that of interleukin-10 in NSCLC/PBMC co-cultures. Taken together, our results suggest that specific knockdown for hnRNP A1/A2 or SF2/ASF increase exclusion of exons 2 and 3 of IRF-3 pre-mRNA and influence immunomodulatory functions of human NSCLC cells.

The altered Schaedler flora (ASF) is a bacterial community that supports normal growth and development of gnotobiotic mice. We report here the draft genome sequences of the 8 bacteria that comprise the ASF.

Variable levels of aberrantly spliced cystic fibrosis transmembrane conductance regulator (CFTR ) transcripts were suggested to correlate with variable cystic fibrosis (CF) severity. We studied the effect of the cellular splicing factors, hnRNP A1 and ASF/SF2, and their adenoviral analogues, E4-ORF6 and E4-ORF3, that promote exon skipping and/or exon inclusion, on the splicing pattern of the CFTR mutation 3849+10kb C->>T and the 5T allele. These mutations can lead to cryptic exon inclusion and exon skipping, respectively. Overexpression of the cellular factors promoted exon skipping of pre-mRNA transcribed from minigenes carrying the mutation (p5T or p3849M). This led to a substantial decrease in the level of correctly spliced mRNA transcribed from p5T and generated correctly spliced mRNA transcribed from p3849M that was not found without overexpression of the factors. The viral factor, E4-ORF3, promoted exon inclusion and led to a substantial increase of the correctly spliced mRNA transcribed from the p5T. The factor, E4-ORF6, activated exon skipping and generated correctly spliced mRNA transcribed from p3849M. Thus, overexpression of alternative splicing factors can modulate the splicing pattern of CFTR alleles carrying splicing mutations. These results are important for understanding the mechanism underlying phenotypic variability in CF and other genetic diseases.

Serine-arginine-rich (SR) proteins, a family of spliceosomal proteins, function at multiple steps in the assembly of the spliceosome in non-plant systems. Limited studies with metazoan SR splicing factors (ASF/SF2 and SC35) indicated that their mobility is not dependent on ATP and phosphorylation. In addition, inhibition of transcription slightly increased their mobility. Here, we analyzed the mobility of SR45, a plant-specific SR protein with unique domain organization, and SR1/SRp34, a plant homolog of metazoan ASF/SF2, using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). Our results show that, in contrast to metazoan SR splicing factors, the movement of the plant SR proteins is dependent on ATP, phosphorylation and transcription. To understand the underlying mechanism for these observations, we carried out mobility analyses with the domain-deletion mutants of SR45 in ATP-depleted cells and in the presence of inhibitors of transcription or phosphorylation. Our results show that the sensitivity of SR45 to these inhibitors is conferred by an RNA-recognition motif (RRM) and the serine-arginine-rich (RS) domain 2. These results provide important insights into the mechanisms of plant SR protein movement and suggest fundamental differences in the regulation of the mobility of plant and animal SR splicing factors.

During the translocation of tRNAs and mRNA relative to the ribosome, the B1a, B1b and B1c bridges undergo the most extensive conformational changes among the bridges between the large and the small ribosomal subunits. The B1a bridge, also called the "A-site finger" (ASF), is formed by the 23S rRNA helix 38, which is located right above the ribosomal A-site. Here, we deleted part of the ASF so that the B1a intersubunit bridge could not be formed (DeltaB1a). The mutation led to a less efficient subunit association. A number of functional activities of the DeltaB1a ribosomes, such as tRNA binding to the P and A-sites, translocation and EF-G-related GTPase reaction were preserved. A moderate decrease in EF-G-related GTPase stimulation by the P-site occupation by deacylated tRNA was observed. This suggests that the B1a bridge is not involved in the most basic steps of the elongation cycle, but rather in the fine-tuning of the ribosomal activity. Chemical probing of ribosomes carrying the ASF truncation revealed structural differences in the 5S rRNA and in the 23S rRNA helices located between the peptidyltransferase center and the binding site of the elongation factors. Interestingly, reactivity changes were found in the P-loop, an important functional region of the 23S rRNA. It is likely that the A-site finger, in addition to its role in subunit association, forms part of the system of allosteric signal exchanges between the small subunit decoding center and the functional centers on the large subunit.

Expression of cytoplasmic mRNA from most adenovirus transcription units is subjected to a temporal regulation at the level of alternative pre-mRNA splicing. The general tendency is that splice site selection changes from proximal to distal late after infection. Interestingly, ASF/SF2, which is a prototypical member of the SR family of splicing factors, has the opposite effect on splice site selection, inducing an increase in proximal splice site usage. We have previously shown that SR proteins late during an adenovirus infection become partially inactivated as splicing regulatory proteins. A prediction from these results is that overexpression of an SR protein, such as ASF/SF2, during virus growth will interfere with virus replication by disturbing the balance of functional and nonfunctional ASF/SF2 in the infected cell. To test this hypothesis, we reconstructed a recombinant adenovirus expressing ASF/SF2 under the transcriptional control of a regulated promoter. The results show that, as predicted, induction of ASF/SF2 during lytic virus growth prevents the early to late shift in mRNA expression from both early (E1A and E1B) and late (L1) transcription units. Furthermore, ASF/SF2 overexpression blocks viral DNA replication and reduces selectively cytoplasmic accumulation of major late mRNA, resulting in a lower virus yield. Collectively, our results provide additional support for the hypothesis that viral control of SR protein function is important for the proper expression of viral proteins during lytic virus growth.

We have analysed the promoter regions of two closely related auxin-regulated glutathione S-transferase genes. All active deletion constructs tested showed expression of the reporter gene beta-glucuronidase (gusA) in root tips of young seedlings and newly developing lateral roots. Auxin treatment greatly enhanced the level of expression. The Nt103-1 promoter region -370/-276 was found to be necessary, at least as a quantitative element to confer auxin-responsiveness to a reporter gene, and sequences responsible for the auxin-responsiveness must be located downstream of -370. The region -651/-370 contains sequence information necessary for uninduced expression. The Nt103-35 promoter manifested its auxin-responsiveness within the -504/-310 region. Electrophoretic mobility shift analysis, using nuclear extracts from tobacco leaves and suspension cells, identified a factor binding to a sequence (ap103, TGAGTCT) at position -560 of the Nt103-1 promoter, which shows homology to the mammalian AP-1 site. A second factor was found to bind a sequence (as103, ATAGCTAAGTGCTTACG) with homology to the CaMV 35S promoter as-1 element. The as103 element is present in both promoters and positioned around -360, so within the region determined to be indispensable for the response to auxin. A third factor was found binding to the -276/-190 region of both promoters. Combined, these data point to the relevance of a 90 bp region for auxin-induced activity of both tobacco genes. The ASF-1 like factor binding to the as103 element within this region might be involved in mediating the auxin response.

Hyperventilation-induced bronchoconstriction (HIB) is a component of exercise-induced asthma (EIA) believed to result from the penetration of unconditioned air into the lung periphery. We used a canine model of EIA to examine the effect of hyperventilation on airway surface fluid (ASF) volume and osmolality, and to determine if the observed kinetics support the hypothesis that hyperventilation-induced changes in ASF osmolality initiate bronchoconstriction. Exposure of sublobar airways to dry air at baseline insufflation resulted in stable measurements of ASF volume, ASF osmolality, and peripheral airway resistance (Rp). Baseline insufflation of warm humidified air increased ASF volume, but did not alter ASF osmolality. Hyperventilation challenge with warm humidified air (WAC) increased Rp and ASF volume, but decreased ASF osmolality. Dry air challenge (DAC) increased Rp, ASF volume, and ASF osmolality. ASF osmolality during DAC was markedly higher when compared with posthyperventilation values. Post-DAC changes in (Delta) ASF volume and osmolality were poorly correlated with the development of HIB. In contrast to Delta ASF after DAC, Delta ASF osmolality during DAC was strongly correlated with HIB, and tended to be inversely related to Delta ASF volume. These observations are consistent with the hypothesis that changes in airway osmolality during hyperventilation initiate peripheral airway constriction.

Alternative splicing regulators have emerged as new players in cancer development, modulating the activities of many tumor suppressors and oncogenes and regulating the signaling pathways. However, little is known about the mechanisms by which these oncogenic splicing factors lead to cellular transformation. We have shown previously that the splicing factor serine and arginine splicing factor 1 (SRSF1; SF2/ASF) is a proto-oncogene, which is amplified in breast cancer and transforms immortal cells when overexpressed. In this study, we performed a structure-function analysis of SRSF1 and found that the RNA recognition motif 1 (RRM1) domain is required for its oncogenic activity. Deletion of RRM1 eliminated the splicing activity of SRSF1 on some of its endogenous targets. Moreover, we found that SRSF1 elevates the expression of B-Raf and activates the mitogen-activated protein kinase kinase (MEK) extracellular signal-regulated kinase (ERK) pathway and that RRM1 is required for this activation as well. B-Raf-MEK-ERK activation by SRSF1 contributes to transformation as pharmacological inhibition of MEK1 inhibits SRSF1-mediated transformation. In conclusion, RRM1 of SRSF1 is both required (and when tethered to the RS domain) also sufficient to activate the Raf-MEK-ERK pathway and to promote cellular transformation.

METHODS

Systematic review.

OBJECTIVE

To review the literature on proximal junctional kyphosis (PJK) as a specific form for proximal adjacent segment pathology and report on the incidence, timing, risk factors, and effect on health-related quality of life (HRQOL) outcomes reported for PJK.

BACKGROUND

PJK is a complication of spinal deformity surgery that can compromise outcomes and necessitate revision surgery. Multiple risk factors have been associated with PJK, making the etiology multifactorial. Knowledge of the risk factors is important for minimizing the occurrence of PJK and to allow surgeons to take measures for its prevention when possible.

METHODS

A systematic search of PubMed, CINAHL, EMBASE, the Cochrane Library, and Google Scholar through February 15, 2012, was performed. The focus was on studies designed to evaluate PJK in patients who had surgery for scoliosis and/or kyphosis. Adjusted effect sizes and significance based on adjusting for confounders were reported if available, otherwise, crude risk ratios and 95% confidence intervals were calculated.

RESULTS

The search yielded 85 citations and 8 met the criteria for inclusion. The incidence of PJK ranged from 17% to 39% and the majority seemed to occur within 2 years of surgery. The most common patient demographic associated with a higher PJK risk was increased age. Surgery-related risk factors were fusions to the sacrum, combined anterior/posterior surgery, thoracoplasty, and upper instrumented vertebra at T1-T3. Postoperative hypokyphosis or hyperkyphosis was associated with an increased risk of PJK. Despite the presence of PJK, health-related quality of life outcomes were not affected.

CONCLUSIONS

Patients at higher risk for PJK are those who are of older age, who had fusions to the sacrum, combined anterior/posterior surgery, thoracoplasty, and an upper instrumented vertebra at T1-T3. Despite the presence of PJK, no differences were noted in health-related quality of life outcomes.

METHODS

1. The risk of developing PJK above a spinal deformity fusion is 17% to 39%, with most noted by 2 years postoperative.

METHODS

Moderate. Strength of Statement: Strong. 2. The risk factors of PJK development include increased age, fusion to sacrum, combined ASF/PSF, thoracoplasty, UIV at T1–T3, and nonanatomic restoration of thoracic kyphosis.

METHODS

Low. Strength of Statement: Weak. 3. The development of PJK does not seem to have a detrimental effect on HRQOL outcomes, at least in milder/nonrevision forms.

METHODS

Moderate. Strength of Statement: Weak.

SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, we monitored region-specific mapping of phosphorylation sites in ASF/SF2 as a function of the protein phosphatase PP1. We showed that 10 phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RNA recognition motifs protects against dephosphorylation, suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.

SR proteins (splicing factors containing arginine-serine repeats) are essential factors that control the splicing of precursor mRNA by regulating multiple steps in spliceosome development. The prototypical SR protein ASF/SF2 (human alternative splicing factor) contains two N-terminal RNA recognition motifs (RRMs) (RRM1 and RRM2) and a 50-residue C-terminal RS (arginine-serine-rich) domain that can be phosphorylated at numerous serines by the protein kinase SR-specific protein kinase (SRPK) 1. The RS domain [C-terminal domain that is rich in arginine-serine repeats (residues 198-248)] is further divided into N-terminal [RS1: N-terminal portion of the RS domain (residues 198-227)] and C-terminal [RS2: C-terminal portion of the RS domain (residues 228-248)] segments whose modification guides the nuclear localization of ASF/SF2. While previous studies revealed that SRPK1 phosphorylates RS1, regiospecific and temporal-specific control within the largely redundant RS domain is not well understood. To address this issue, we performed engineered footprinting and single-turnover experiments to determine where and how SRPK1 initiates phosphorylation within the RS domain. The data show that local sequence elements in the RS domain control the strong kinetic preference for RS1 phosphorylation. SRPK1 initiates phosphorylation in a small region of serines (initiation box) in the middle of the RS domain at the C-terminal end of RS1 and then proceeds in an N-terminal direction. This initiation process requires both a viable docking groove in the large lobe of SRPK1 and one RRM (RRM2) on the N-terminal flank of the RS domain. Thus, while local RS/SR content steers regional preferences in the RS domain, distal contacts with SRPK1 guide initiation and directional phosphorylation within these regions.

Autosomal dominant vitreoretinochoroidopathy (ADVIRC), a retinal dystrophy often associated with glaucoma and cataract, forms part of a phenotypic spectrum of 'bestrophinopathies'. It has been shown previously that ADVIRC results from BEST1 mutations that cause exon skipping and lead to the production of shortened and internally deleted isoforms. This study describes a novel ADVIRC mutation and show that it disrupts an exonic splice enhancer (ESE) site, altering the binding of a splicing-associated SR protein. As with previous ADVIRC mutations, the novel c.704T->>C mutation in exon 6 altered normal splicing in an ex vivo splicing assay. Both this and another exon 6 ADVIRC-causing mutation (c.707G->>A) either weakened or abolished splicing in an ESE-dependent splice assay compared with a nearby exon 6 mutation associated with Best disease (c.703G->>C). Gel shift assays were undertaken with RNA oligonucleotides encompassing the ADVIRC and Best disease mutations with four of the most commonly investigated SR proteins. Although SC35, SRp40 and SRp55 proteins all bound to the wild-type and mutated sequences with similar intensities, there was increased binding of ASF/SF2 to the two ADVIRC-mutated sequences compared with the wild-type or Best disease-mutated sequences. The exon skipping seen for these two exon 6 ADVIRC mutations and their affinity for ASF/SF2 suggests that the region encompassing these mutations may form part of a CERES (composite exonic regulatory elements of splicing) site.

Rous sarcoma virus (RSV) requires large amounts of unspliced RNA for replication. Splicing and polyadenylation are coupled in the cells they infect, which raises the question of how viral RNA is efficiently polyadenylated in the absence of splicing. Optimal RSV polyadenylation requires a far-upstream splicing control element, the negative regulator of splicing (NRS), that binds SR proteins and U1/U11 snRNPs and functions as a pseudo-5' splice site that interacts with and sequesters 3' splice sites. We investigated a link between NRS-mediated splicing inhibition and efficient polyadenylation. In vitro, the NRS alone activated a model RSV polyadenylation substrate, and while the effect did not require the snRNP-binding sites or a downstream 3' splice site, SR proteins were sufficient to stimulate polyadenylation. Consistent with this, SELEX-binding sites for the SR proteins ASF/SF2, 9G8, and SRp20 were able to stimulate polyadenylation when placed upstream of the RSV poly(A) site. In vivo, however, the SELEX sites improved polyadenylation in proviral clones only when the NRS-3' splice site complex could form. Deletions that positioned the SR protein-binding sites closer to the poly(A) site eliminated the requirement for the NRS-3' splice site interaction. This indicates a novel role for SR proteins in promoting RSV polyadenylation in the context of the NRS-3' splice site complex, which is thought to bridge the long distance between the NRS and poly(A) site. The results further suggest a more general role for SR proteins in polyadenylation of cellular mRNAs.