ORMOCHALCs: organically modified chalcogenide polymers for infrared optics.

A novel method combining elemental sulfur and selenium was developed, yielding crystalline sulfur-selenium compounds. The compounds were melted, and an organic comonomer added. Once the organic comonomer was consumed, the viscous compound was vitrified and allowed to cool yielding organic-inorganic hybrid polymers that are termed Organically Modified Chalcogenide (ORMOCHALC) polymers.

Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis.

p63 is critical for squamous development and exists as multiple isotypes of two subclasses, TA and DeltaN. DeltaNp63 isotypes can antagonize transcription by TAp63 and p53, and are highly expressed in squamous cell cancers. Using mouse keratinocytes as a biological model of squamous epithelium, we show that multiple p63 isotypes, DeltaN- and TA-containing, are expressed and differentially modulated during in vitro murine keratinocyte differentiation. DeltaNp63alpha declines with Ca2+-induced differentiation, while a smaller DeltaN-form, DeltaNp63s, persists, suggesting unique functions of the two DeltaN-forms. To investigate the impact of dysregulated p63 expression that is observed in cancers and to define the biological contribution of the different domains of the p63 isotypes, DeltaNp63alpha, DeltaNp63p40, TAp63alpha, TAp63gamma or beta-galactosidase were overexpressed in primary murine keratinocytes. Microarray, RT-PCR and western blot analyses revealed that overexpression of DeltaNp63p40, which lacks the entire alpha-tail present in DeltaNp63alpha, permits expression of a full panel of differentiation markers. This is in contrast to overexpression of the full-length DeltaNp63alpha, which blocks induction of keratin 10, loricrin and filaggrin. These findings support a role for the alpha-tail of DeltaNp63alpha in blocking differentiation-specific gene expression. Overexpression of either TAp63 isotype permits keratin 10 and loricrin expression, thus the alpha-terminus requires the cooperation of the DeltaN domain in blocking early differentiation. However, both TA isotypes block filaggrin induction. The DeltaN-terminus is sufficient to maintain keratinocytes in a proliferative state, as both DeltaN forms block Ca2+-mediated p21WAF1 induction and S-phase arrest, while sustaining elevated PCNA levels. No alteration in cell cycle regulation was observed in keratinocytes overexpressing TAp63alpha or TAp63gamma. Clarifying the functional distinctions between p63 isotypes and domains will help to elucidate how their dysregulation impacts tumor biology and may suggest novel therapeutic strategies for modulating behavior of tumor cells with altered expression of p53 family members.

Selective aortic arch perfusion with hemoglobin-based oxygen carrier-201 for resuscitation from exsanguinating cardiac arrest in swine.

OBJECTIVE: The prospects for resuscitation after blunt traumatic cardiac arrest are dismal. Selective aortic arch perfusion (SAAP) with a hemoglobin-based oxygen carrier (HBOC-201) offers a potentially effective therapy. This study evaluated the acute cardiovascular and metabolic effects of SAAP with HBOC-201 in an exsanguination model of cardiac arrest. DESIGN: Randomized, controlled, laboratory investigation. SETTING: University research laboratory. SUBJECTS: Domestic swine, 25-39 kg. INTERVENTIONS: Partial resection of four liver lobes rapidly led to profound hemorrhagic shock and subsequent cardiac arrest at 10-13 mins. At 15 mins, swine were randomized to receive either SAAP with oxygenated lactated Ringer's (LR) solution (n = 6) or SAAP with oxygenated HBOC-201 (n = 6) at a rate of 10 mL x kg(-1) x min(-1) until return of spontaneous circulation with a mean aortic pressure of 60 mm Hg (8.0 kPa) was achieved. Epinephrine (0.005 mg/kg) was given via intra-aortic route every 30 secs as needed to promote return of spontaneous circulation beginning at 18 mins after onset of liver injury (3 mins after beginning SAAP). MEASUREMENTS AND MAIN RESULTS: Mean aortic pressure, cardiac output, total blood loss, and time of arrest were similar for both groups before SAAP therapy. In the SAAP-HBOC group, return of spontaneous circulation with a sustained mean aortic pressure of 60 mm Hg (8.0 kPa) was achieved in six of six swine at 1.9 +/- 0.3 mins of SAAP, and none of these swine required epinephrine. In the SAAP-LR group, no swine (from a total of six) achieved return of spontaneous circulation before intra-aortic epinephrine administration, and only two of six swine had brief return of spontaneous circulation with an mean aortic pressure of 60 mm Hg (8.0 kPa) after intra-aortic epinephrine that was sustained for <10 mins. One-hour survival was five of six in the SAAP-HBOC group and none of six in the SAAP-LR group (p <.05, Fisher's exact test). CONCLUSION: SAAP with oxygenated HBOC-201 rapidly restored viable cardiovascular function after exsanguinating cardiac arrest in this swine model of liver injury with profound hemorrhagic shock.

Messenger RNA repair and restoration of protein function by spliceosome-mediated RNA trans-splicing.

The functional repertoire of the human genome is amplified by the differential assortment of exons. Spliceosome-mediated RNA trans-splicing can mobilize these packets of genetic information to reprogram mRNAs. In principle, this process could repair defective transcripts in loss-of-function genetic disorders in humans. We developed a tractable lacZ repair system to serve as a model for these genetic disorders. Targeted pre-trans-splicing RNA molecules efficiently and specifically repaired mutated lacZ transcripts and restored enzymatic activity in human cells. The development of this model confirms the potential for spliceosome-mediated RNA trans-splicing in genetic repairs and provides a powerful tool for rational design and in vitro evolution of pre-trans-splicing molecules.

The 3'-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo.

We describe a new approach to elucidate the role of 3'-end processing in pre-mRNA splicing in vivo using the influenza virus NS1A protein. The effector domain of the NS1A protein, which inhibits the function of the CPSF and PABII factors of the cellular 3'-end-processing machinery, is sufficient for the inhibition of not only 3'-end formation but also the splicing of single-intron pre-mRNAs in vivo. We demonstrate that inhibition of the splicing of single-intron pre-mRNAs results from inhibition of 3'-end processing, thereby establishing that 3'-end processing is required for the splicing of a 3' terminal intron in vivo. Because the NS1A protein causes a global suppression of 3'-end processing in trans, we avoid the ambiguities caused by the activation of cryptic poly(A) sites that occurs when mutations are introduced into the AAUAAA sequence in the pre-mRNA. In addition, this strategy enabled us to establish that the function of a particular 3'-end-processing factor, namely CPSF, is required for the splicing of single-intron pre-mRNAs in vivo: splicing is inhibited only when the effector domain of the NS1A protein binds and inhibits the function of the 30-kDa CPSF protein in 3'-end formation. In contrast, the 3'-end processing factor PABII is not required for splicing. We discuss the implications of these results for cellular and influenza viral mRNA splicing.

Alteration in Kupffer cell function after mild hemorrhagic shock.

Functional changes in Kupffer cells occur after profound hemorrhagic shock. This study was performed to demonstrate if Kupffer cell changes also occur after mild hemorrhagic shock. Sprague-Dawley rats were bled to a systolic blood pressure of 60 to 70 mmHg and resuscitated with Lactated Ringers solution (twice the shed blood volume) after 30 min. Resuscitation produced immediate recovery of blood pressure and allowed long-term recovery of the animals. Sham animals received anesthesia and monitoring only. Thirty minutes after resuscitation, Kupffer cells were isolated by centrifugal elutriation and cultured for 48 h. In Kupffer cells isolated from shocked animals, phorbol ester-stimulated superoxide production increased 7-fold and lipopolysaccharide- (LPS) stimulated prostaglandin E2 (PGE2) production increased 4-fold. Tumor necrosis factor-alpha (TNFalpha) production, on the other hand, was decreased by 50%. A non-significant trend toward increased phagocytosis was also observed, whereas LPS-stimulated nitric oxide production was unchanged. In conclusion, mild hemorrhagic shock produced increases in superoxide and PGE2 production, and decreases in TNFalpha production by Kupffer cells, changes that may be appropriate to defend against the infectious challenges that often follows trauma and hemorrhage.

An objective analysis of process errors in trauma resuscitations.

OBJECTIVE: A computer-based system to apply trauma resuscitation protocols to patients with penetrating thoracoabdominal trauma was previously validated for 97 consecutive patients at a Level 1 trauma center by a panel of the trauma attendings and further refined by a panel of national trauma experts. The purpose of this article is to describe how this system is now used to objectively critique the actual care given to those patients for process errors in reasoning, independent of outcome. METHODS: A chronological narrative of the care of each patient was presented to the computer program. The actual care was compared with the validated computer protocols at each decision point and differences were classified by a predetermined scoring system from 0 to 100, based on the potential impact on outcome, as critical/noncritical/no errors of commission, omission, or procedure selection. RESULTS: Errors in reasoning occurred in 100% of the 97 cases studied, averaging 11.9/case. Errors of omission were more prevalent than errors of commission (2. 4 errors/case vs 1.2) and were of greater severity (19.4/error vs 5. 1). The largest number of errors involved the failure to record, and perhaps observe, beside information relevant to the reasoning process, an average of 7.4 missing items/patient. Only 2 of the 10 adverse outcomes were judged to be potentially related to errors of reasoning. CONCLUSIONS: Process errors in reasoning were ubiquitous, occurring in every case, although they were infrequently judged to be potentially related to an adverse outcome. Errors of omission were assessed to be more severe. The most common error was failure to consider, or document, available relevant information in the selection of appropriate care.

Proinflammatory kupffer cell alterations after femur fracture trauma and sepsis in rats.

This study examined effects of trauma and sepsis on Kupffer cell function. When CBA/J mice had femur fracture (FFx), no deaths occurred. After cecal ligation and puncture (CLP), 44% died. Following combined injuries (FFx + CLP), mortality increased to 60%, suggesting a deleterious effect between FFx + CLP. Kupffer cell ablation with GdCI3 decreased mortality to 13% after CLP and 5% after FFx + CLP. After FFx, CLP, and FFx + CLP, Kupffer cells isolated from Sprague-Dawley rats produced 720%, 1,100%, and 2,130% more O2. than sham, respectively. Phagocytosis increased 320%, 610%, and 150%. Kupffer cell PGE2 production also increased 300%, 510%, and 300% over sham. After FFx alone, TNF-alpha production decreased 40%. By contrast, CLP and FFx + CLP increased TNF-alpha release 25% and 100%, respectively. After FFx, NO. production decreased 44%, whereas NO increased 280% and 260% after CLP and FFx + CLP. These findings indicate that Kupffer cells mediate mortality after CLP and FFx + CLP. Increased mortality is associated with a more proinflammatory and less antimicrobial Kupffer cell phenotype.

Accuracy of administrative data in trauma: splenic injuries as an example.

BACKGROUND: Accurate data are needed to evaluate clinical outcomes, therapeutic modalities, and quality of care in trauma. Administrative data, usually used for billing, have been used to evaluate performance and assess therapy in other medical specialties. This study was performed to determine whether administrative databases are accurate in the recording of information about trauma patients with splenic injuries. METHODS: Patients who had blunt splenic injuries were identified using a state trauma registry. The medical records of those patients were reviewed. The data collected by chart review were compared with data in the statewide administrative database of patients who had splenic injuries at the same four Level I and II trauma centers in the same 5-year period. Age, sex, admission date, and hospital were matched to assure comparison of the identical cohort. chi2 analysis was used to compare dichotomous data and Student's t test continuous data. RESULTS: The administrative database identified 641 and the trauma registry identified 529 patients with a diagnosis of splenic injury. A total of 401 patients were found in both databases. Of these, 120 (22.7%) patients were not recorded in the administrative database. Injury Severity Score was underreported by the administrative database (25.74 +/- 14.7 vs. 19.52 +/- 11, p < 0.0001). The administrative database underreported orthopedic, chest, and head injuries (317 vs. 215, 325 vs. 228, and 234 vs. 155, respectively; all p < 0.0001). Use of abdominal computed tomographic scan and diagnostic peritoneal lavage were also underreported (260 vs. 56 and 104 vs.17, both p < 0.0001). The number of operations on the spleen and number of orthopedic procedures were underreported (259 vs. 225, p < 0.014 and 147 vs. 94, p < 0.0001). Complications were markedly underreported by the administrative database (200 vs. 47, p < 0.0001) CONCLUSION: This study shows that administrative data lack accuracy in the recording of associated injuries, injury severity, diagnostics, procedures, and outcomes data in patients with splenic injuries. Whether these data should be used to evaluate treatment modalities or quality of care in trauma is questionable.

Utilization of the bovine papillomavirus type 1 late-stage-specific nucleotide 3605 3' splice site is modulated by a novel exonic bipartite regulator but not by an intronic purine-rich element.

Bovine papillomavirus type 1 (BPV-1) late gene expression is regulated at both transcriptional and posttranscriptional levels. Maturation of the capsid protein (L1) pre-mRNA requires a switch in 3' splice site utilization. This switch involves activation of the nucleotide (nt) 3605 3' splice site, which is utilized only in fully differentiated keratinocytes during late stages of the virus life cycle. Our previous studies of the mechanisms that regulate BPV-1 alternative splicing identified three cis-acting elements between these two splice sites. Two purine-rich exonic splicing enhancers, SE1 and SE2, are essential for preferential utilization of the nt 3225 3' splice site at early stages of the virus life cycle. Another cis-acting element, exonic splicing suppressor 1 (ESS1), represses use of the nt 3225 3' splice site. In the present study, we investigated the late-stage-specific nt 3605 3' splice site and showed that it has suboptimal features characterized by a nonconsensus branch point sequence and a weak polypyrimidine track with interspersed purines. In vitro and in vivo experiments showed that utilization of the nt 3605 3' splice site was not affected by SE2, which is intronically located with respect to the nt 3605 3' splice site. The intronic location and sequence composition of SE2 are similar to those of the adenovirus IIIa repressor element, which has been shown to inhibit use of a downstream 3' splice site. Further studies demonstrated that the nt 3605 3' splice site is controlled by a novel exonic bipartite element consisting of an AC-rich exonic splicing enhancer (SE4) and an exonic splicing suppressor (ESS2) with a UGGU motif. Functionally, this newly identified bipartite element resembles the bipartite element composed of SE1 and ESS1. SE4 also functions on a heterologous 3' splice site. In contrast, ESS2 functions as an exonic splicing suppressor only in a 3'-splice-site-specific and enhancer-specific manner. Our data indicate that BPV-1 splicing regulation is very complex and is likely to be controlled by multiple splicing factors during keratinocyte differentiation.

Optimization of a weak 3' splice site counteracts the function of a bovine papillomavirus type 1 exonic splicing suppressor in vitro and in vivo.

Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3' splice site utilization from an early 3' splice site at nucleotide (nt) 3225 to a late-specific 3' splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3' splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3' splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3' splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3' splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3' splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3' splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3' splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3' splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3' splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3' splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

Production of infectious bovine papillomavirus from cloned viral DNA by using an organotypic raft/xenograft technique.

Bovine papillomavirus type 1 (BPV-1) induces fibropapillomas in its natural host and can transform fibroblasts in culture. The viral genome is maintained as an episome within fibroblasts, which has allowed extensive genetic analyses of the viral functions required for DNA replication, gene expression, and transformation. Much less is known about BPV-1 gene expression and replication in bovine epithelial cells because the study of the complete viral life cycle requires an experimental system capable of generating a fully differentiated stratified bovine epithelium. Using a combination of organotypic raft cultures and xenografts on nude mice, we have developed a system in which BPV-1 can replicate and produce infectious viral particles. Organotypic cultures were established with bovine keratinocytes plated on a collagen raft containing BPV-1-transformed fibroblasts. These keratinocytes were infected with virus particles isolated from a bovine wart or were transfected with cloned BPV-1 DNA. Several days after the rafts were lifted to the air interface, they were grafted on nude mice. After 6-8 weeks, large xenografts were produced that exhibited a hyperplastic and hyperkeratotic epithelium overlying a large dermal fibroma. These lesions were strikingly similar to a fibropapilloma caused by BPV-1 in the natural host. Amplified viral DNA and capsid antigens were detected in the suprabasal cells of the epithelium. Moreover, infectious virus particles could be isolated from these lesions and quantitated by a focus formation assay on mouse cells in culture. Interestingly, analysis of grafts produced with infected and uninfected fibroblasts indicated that the fibroma component was not required for productive infection or morphological changes characteristic of papillomavirus-infected epithelium. This system will be a powerful tool for the genetic analysis of the roles of the viral gene products in the complete viral life cycle.

Relatively short diagnostic delays (<8 hours) produce morbidity and mortality in blunt small bowel injury: an analysis of time to operative intervention in 198 patients from a multicenter experience.

OBJECTIVE: Blunt small bowel injury (SBI) is uncommon, and its timely diagnosis may be difficult. The impact of operative delays on morbidity and mortality has been unclear. The purpose of this study was to determine the relationship of diagnostic delays to morbidity and mortality in blunt SBI. METHODS: Patients with blunt SBI with perforation were identified from the registries of eight trauma centers (1989-1997). Patients with duodenal injuries were excluded. Data were extracted by individual chart review. Patients were classified as multi-trauma (group 1) or near-isolated SBI (group 2 with Abbreviated Injury Scale score < 2 for other body areas). Time to operation and its impact on mortality and morbidity was determined for each patient. RESULTS: A total of 198 patients met inclusion criteria: 66.2% were male, mean age was 35.2 years (range, 1-90 years) and mean Injury Severity Score was 16.7 (range, 9-47). 100 patients had multiple injuries (group 1). There were 21 deaths (10.6%) with 9 (4.5%) attributable to delay in operation for SBI. In patients with near-isolated SBI, the incidence of mortality increased with time to operative intervention (within 8 hours: 2%; 8-16 hours: 9.1%; 16-24 hours: 16.7%; greater than 24 hours: 30.8%, p = 0.009) as did the incidence of complications. Delays as short as 8 hours 5 minutes and 11 hours 15 minutes were associated with mortality attributable to SBI. The rates of delay in diagnosis were not significantly associated with age, gender, intoxication, transfer status, or presence of associated injuries. CONCLUSION: Delays in the diagnosis of SBI are directly responsible for almost half the deaths in this series. Even relatively brief delays (as little as 8 hours) result in morbidity and mortality directly attributable to "missed" SBI. Further investigation into the prompt diagnosis of this injury is needed.

Parameters that affect in vitro splicing of bovine papillomavirus type 1 late pre-mRNAs.

During the course of study on regulated viral pre-mRNA splicing, an in vitro RNA splicing assay was developed to analyze how an exonic splicing enhancer stimulates splicing of bovine papillomavirus type 1 (BPV-1) late pre-mRNAs. The optimal concentration of HeLa nuclear extract (HNE) in a standard RNA splicing reaction depends on the individual substrate pre-mRNA. Splicing of a BPV-1 late pre-mRNA required 40% HNE and 2 h incubation at 30 degrees C. Higher HNE was detrimental to splicing and longer incubation times lead to RNA degradation. In the reaction containing 40% EDTA-treated HNE, 1.5-3.0 mM Mg2+ or 3 mM Mn2+, but not Co2+, were required to catalyze an efficient splicing reaction. Surprisingly, EDTA-untreated HNE in the absence of exogenous Mg2+ catalyzed very efficiently splicing of the RNA. Addition of Mg2+ from 0.1 to 0.5 mM, only enhanced slightly the splicing in EDTA-untreated HNE and excessive Mg2+ concentration (above 1.5 mM) in the reaction resulted in production of aberrant splicing products or intermediates. In contrast, addition of Mn2+ to EDTA-untreated HNE severely suppressed splicing. In addition, it was observed that the RNA transcribed from vector sequences downstream of the polylinker region of pSP72 vector, when connected to the 3' terminus of chimeric Drosophila doublesex-BPV-1 SE1 pre-mRNAs, suppressed dramatically splicing. RNA transcribed from the pSP72 polylinker region, when supplied in trans, also suppressed splicing. These results suggest that a DNA template used to make RNA transcripts should avoid these sequences as much as possible.

Bovine hemoglobin-based oxygen carrier (HBOC-201) for resuscitation of uncontrolled, exsanguinating liver injury in swine. Carolina Resuscitation Research Group.

In the setting of rapidly exsanguinating hemorrhage, resuscitation with intravenous (i.v.) crystalloid solution may not sustain survival before availability of allogenic blood transfusion and surgery. This study tested the hypothesis that bovine hemoglobin-based oxygen carrier, HBOC-201, would improve resuscitation and extend early survival from exsanguinating hemorrhage. This study simulated the prehospital scenario of rapidly exsanguinating hemorrhage with prolonged prehospital time and lack of blood availability. Severe hemorrhagic shock was induced in swine by using multiple liver lacerations. At 9 min after the onset of bleeding, swine were randomized to receive approximately 10 mL/kg/min of i.v. lactated Ringer's solution (n = 10) or HBOC-201 (n = 7) to achieve a mean aortic pressure (MAP) of 60 mmHg. Thereafter, infusion rate was adjusted to maintain MAP at 60 mmHg for up to 2 h. All animals were initially successfully resuscitated. The results showed 2-h survival was 1 of 10 with lactated Ringer's and 7 of 7 with HBOC-201 (P = 0.0004). Nine lactated Ringer's swine had cardiovascular collapse at 36 +/- 10 min. Lactate at 30 min was 18 +/- 3 mmol/L with lactated Ringer's and 12 +/- 2 mmol/L with HBOC-201 (P < 0.05). Hematocrit was <1% in 9 of 10 lactated Ringer's and 6 of 7 HBOC-201 animals. These data indicate that HBOC-201 improved early survival and stabilized hemodynamic and metabolic parameters vs. lactated Ringer's in this swine model of liver injury with uncontrolled, lethal hemorrhage that simulates the prehospital care environment where allogenic blood is unavailable.

Accuracy of administrative data in trauma.

BACKGROUND: Accurate data are needed to evaluate outcomes, therapeutics, and quality of care. This study assesses the accuracy of administrative databases in recording information about trauma patients. METHODS: Patients with thoracic aorta injury were identified with a state trauma registry, and the medical records were reviewed. Data collected were compared to administrative data on patients with thoracic aorta injuries, at the same hospitals in the same time period. RESULTS: Fifteen patients (16.3%) with thoracic aorta injury were not recorded in the administrative database, and 23 patients (18.7%) were misdiagnosed. Ninety-one patients were found in both data sources. The administrative database significantly (P < .05) underrecorded abdominal injuries (50 vs 35), orthopedic injuries (117 vs 75), and chest injuries (77 vs 48). The number of aortograms (78 vs 8), type of operative procedures (use of graft; 70 vs 30), use of bypass (35 vs 16), and complications (77 vs 33) were underreported (P < .05). The Injury Severity Score was underestimated by the administrative database (38.65 +/- 12.41 vs 25.66 +/- 9.53; P < .05). CONCLUSIONS: Administrative data lack accuracy in the recording of associated injury, injury severity, diagnostic, and procedural data. Whether these data should be used to evaluate treatment or quality of care in trauma is questionable.

Function of a bovine papillomavirus type 1 exonic splicing suppressor requires a suboptimal upstream 3' splice site.

Alternative splicing is an important mechanism for the regulation of bovine papillomavirus type 1 (BPV-1) gene expression during the virus life cycle. Previous studies in our laboratory have identified two purine-rich exonic splicing enhancers (ESEs), SE1 and SE2, located between two alternative 3' splice sites at nucleotide (nt) 3225 and nt 3605. Further analysis of BPV-1 late-pre-mRNA splicing in vitro revealed a 48-nt pyrimidine-rich region immediately downstream of SE1 that inhibits utilization of the nt 3225 3' splice site. This inhibitory element, which we named an exonic splicing suppressor (ESS), has a U-rich 5' end, a C-rich central part, and an AG-rich 3' end (Z. M. Zheng, P. He, and C. C. Baker, J. Virol. 70:4691-4699, 1996). The present study utilized in vitro splicing of both homologous and heterologous pre-mRNAs to further characterize the ESS. The BPV-1 ESS was inserted downstream of the 3' splice site in the BPV-1 late pre-mRNA, Rous sarcoma virus src pre-mRNA, human immunodeficiency virus tat-rev pre-mRNA, and Drosophila dsx pre-mRNA, all containing a suboptimal 3' splice site, and in the human beta-globin pre-mRNA, which contains a constitutive 3' splice site. These studies demonstrated that suppression of splicing by the BPV-1 ESS requires an upstream suboptimal 3' splice site but not an upstream ESE. Furthermore, the ESS functions when located either upstream or downstream of BPV-1 SE1. Mutational analyses demonstrated that the function of the ESS is sequence dependent and that only the C-rich region of the ESS is essential for suppression of splicing in all the pre-mRNAs tested.

A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly.

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3' splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3' splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5' region binds U2AF65 and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54-55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3' end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.