Functional implications of novel human acid sphingomyelinase splice variants

Background

Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity.

Methodology/Principal Findings

We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models.

Conclusions/Significance

These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity.     

Conserved sequences in the final intron of MDM2 are essential for the regulation of alternative splicing of MDM2 in response to stress

Alternative splicing plays a fundamental role in generating proteome diversity and is critical in regulation of eukaryotic gene expression. It is estimated that 50% of disease-causing mutations alter splicing efficiency and/or patterns of splicing. An alternatively spliced form of murine double-minute 2, MDM2-ALT1, is associated with pediatric rhabdomyosarcoma (RMS) at high frequency in primary human tumors and RMS cell lines. We have identified that this isoform can be induced in response to specific types of stress (UV and cisplatin). However, the mechanism of alternative splicing of MDM2 in human cancer is unknown. Using UV and cisplatin to model alternative splicing of the MDM2 gene, we have developed a damage-inducible in vitro splicing system. This system employs an MDM2 minigene that mimics the damage-induced alternative splicing observed in vivo. Using this in vitro splicing system, we have shown that conserved intronic sequences in intron 11 of MDM2 are required for normal splicing. Furthermore, we showed that these intronic elements are also required for the regulated damage-induced alternative splicing of MDM2. The use of this novel damage-inducible system will allow for the systematic identification of regulatory elements and factors involved in the splicing regulation of the MDM2 gene in response to stress. This study has implications for identification of novel intervention points for development of future therapeutics for rhabdomyosarcoma.     

Acid Sphingomyelinase Serum Activity Predicts Mortality in Intensive Care Unit Patients after Systemic Inflammation: A Prospective Cohort Study

Introduction

Acid sphingomyelinase is involved in lipid signalling pathways and regulation of apoptosis by the generation of ceramide and plays an important role during the host response to infectious stimuli. It thus has the potential to be used as a novel diagnostic marker in the management of critically ill patients. The objective of our study was to evaluate acid sphingomyelinase serum activity (ASM) as a diagnostic and prognostic marker in a mixed intensive care unit population before, during, and after systemic inflammation.

Methods

40 patients admitted to the intensive care unit at risk for developing systemic inflammation (defined as systemic inflammatory response syndrome plus a significant procalcitonin [PCT] increase) were included. ASM was analysed on ICU admission, before (PCT_before), during (PCT_peak) and after (PCT_low) onset of SIRS. Patients undergoing elective surgery served as control (N = 8). Receiver-operating characteristics curves were computed.

Results

ASM significantly increased after surgery in the eight control patients. Patients from the intensive care unit had significantly higher ASM on admission than control patients after surgery. 19 out of 40 patients admitted to the intensive care unit developed systemic inflammation and 21 did not, with no differences in ASM between these two groups on admission. In patients with SIRS and PCT peak, ASM between admission and PCT_before was not different, but further increased at PCT_peak in non-survivors and was significantly higher at PCT_low compared to survivors. Survivors exhibited decreased ASM at PCT_peak and PCT_low. Receiver operating curve analysis on discrimination of ICU mortality showed an area under the curve of 0.79 for ASM at PCT_low.

Conclusions

In summary, ASM was generally higher in patients admitted to the intensive care unit compared to patients undergoing uncomplicated surgery. ASM did not indicate onset of systemic inflammation. In contrast to PCT however, it remained high in non-surviving ICU patients after systemic inflammation.     

Quantitative Profiling of Drosophila melanogaster Dscam1 Isoforms Reveals No Changes in Splicing after Bacterial Exposure

The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens.     

Splicing of human chloride channel 1

Expression of chloride channel 1 (CLCN1/ClC-1) in skeletal muscle is driven by alternative splicing, a process regulated in part by RNA-binding protein families MBNL and CELF. Aberrant splicing of CLCN1 produces many mRNAs, which were translated into inactive proteins, resulting in myotonia in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. This increase in abnormal splicing variants containing exons 6B, 7A or the insertion of a TAG stop codon just before exon 7 leads to a decrease in expression of the normal splice pattern. The majority of studies examining splicing in CLCN1 have been performed using mouse Clcn1, as have investigations into the activation and suppression of normal splicing variant expression by MBNL1-3 and CELF3–6, respectively. In contrast, examinations of human CLCN1 have been less common due to the greater complexity of splicing patterns. Here, we constructed a minigene containing CLCN1 exons 5–7 and established a novel assay system to quantify the expression of the normal splicing variant of CLCN1 using real-time RT-PCR. Antisense oligonucleotides could promote normal CLCN1 alternative splicing but the effective sequence was different from that of Clcn1. This result differs from previous reports using Clcn1, highlighting the effect of differences in splicing patterns between mice and humans.     

Improvement in Lipid and Protein Trafficking in Niemann‐Pick C1 Cells by Correction of a Secondary Enzyme Defect

Different primary lysosomal trafficking defects lead to common alterations in lipid trafficking, suggesting cooperative interactions among lysosomal lipids. However, cellular analysis of the functional consequences of this phenomenon is lacking. As a test case, we studied cells with defective Niemann‐Pick C1 (NPC1) protein, a cholesterol trafficking protein whose defect gives rise to lysosomal accumulation of cholesterol and other lipids, leading to NPC disease. NPC1 cells also develop a secondary defect in acid sphingomyelinase (SMase) activity despite a normal acid SMase gene (SMPD1). When acid SMase activity was restored to normal levels in NPC1‐deficient CHO cells through SMPD1 transfection, there was a dramatic reduction in lysosomal cholesterol. Two other defects, excess lysosomal bis‐(monoacylglycerol) phosphate (BMP) and defective transferrin receptor (TfR) recycling, were also markedly improved. To test its relevance in human cells, the acid SMase activity defect in fibroblasts from NPC1 patients was corrected by SMPD1 transfection or acid SMase enzyme replacement. Both treatments resulted in a dramatic reduction in lysosomal cholesterol. These data show that correcting one aspect of a complex lysosomal lipid storage disease can reduce the cellular consequences even if the primary genetic defect is not corrected.     

Mapping Allele-Specific DNA Methylation: A New Tool for Maximizing Information from GWAS

In this issue of The Journal, an article by Schalkwyk et al.1 shows the landscape of allele-specific DNA methylation (ASM) in the human genome. ASM has long been studied as a hallmark of imprinted genes, and a chromosome-wide version of this phenomenon occurs, in a random fashion, during X chromosome inactivation in female cells. But the type of ASM motivating the study by Schalkwyk et al. is different. They used a high-resolution, methylation-sensitive SNP array (MSNP) method for genome-wide profiling of ASM in total peripheral-blood leukocytes (PBL) and buccal cells from a series of monozygotic twin pairs. Their data bring a new level of detail to our knowledge of a newly recognized phenomenon—nonimprinted, sequence-dependent ASM. They document the widespread occurrence of this phenomenon among human genes and discuss its basic implications for gene regulation and genetic-epigenetic interactions. But this paper and recent work from other laboratories2,3 raises the possibility of a more immediate and practical application for ASM mapping, namely to help extract maximum information from genome-wide association studies.     

Analysis of the sphingomyelin phosphodiesterase 1 gene (SMPD1) in Turkish Niemann–Pick disease patients: Mutation profile and description of a novel mutation

Niemann–Pick disease (NPD) is a lysosomal storage disorder that results from the deficiency of a lysosomal enzyme, acid sphingomyelinase. Niemann–Pick disease type A and B is caused by mutations in the sphingomyelin phosphodiesterase gene (SMPD1) coding for ASM. The aim of this study was to evaluate the spectrum of SMPD1 gene mutations in Turkish NPD patients and to study genotype–phenotype associations. We present a molecular analysis of 10 Turkish NPD type A/B patients. Four of the patients had type A and six had type B NPD. All mutant SMPD1 alleles were identified, including 5 different mutations, 1 of which was novel. These mutations included three missense mutations: c.409T>C (p.L137P), c.1262 A>G (p.H421R) and c.1552T>C (p.L549P), a common frameshift mutation in codon 189, identified in three patients, is caused by the deletion of the 567T, introducing a stop codon 65 amino acids downstream (p.P189fsX65), and a novel frameshift mutation c.1755delC (p.P585PfsX24) which was not reported previously.     

The Impact of HIV Co-Infection on the Genomic Response to Sepsis

HIV patients have an increased risk to develop sepsis and HIV infection affects several components of the immune system involved in sepsis pathogenesis. We hypothesized that HIV infection might aggrevate the aberrant immune response during sepsis, so we aimed to determine the impact of HIV infection on the genomic host response to sepsis. We compared whole blood leukocyte gene expression profiles among sepsis patients with or without HIV co-infection in the intensive care unit (ICU) and validated our findings in a cohort of patients admitted to the same ICUs in a different time frame. To examine the influence of HIV infection per se, we also determined the expression of genes of interest in a cohort of asymptomatic HIV patients. We identified a predominantly common host response in sepsis patients with or without HIV co-infection. HIV positive sepsis patients in both ICU cohorts showed overexpression of genes involved in granzyme signaling (GZMA, GZMB), cytotoxic T-cell signaling (CD8A, CD8B) and T-cell inhibitory signaling (LAG3), compared to HIV negative patients. Enhanced expression of CD8A, CD8B and LAG3 was also unmasked in asymptomatic HIV patients. Plasma levels of granzymes in sepsis patients were largely below detection limit, without differences according to HIV status. These results demonstrate that sepsis is characterized by a massive common response with few differences between HIV positive and HIV negative sepsis patients. Observed differences in granzyme signaling, cytotoxic T-cell signaling and T-cell inhibitory signaling appear to be changes commonly observed in asymptomatic HIV patients which persist during sepsis.     

Co-regulation of alternative splicing by diverse splicing factors in Caenorhabditis elegans

Regulation of alternative splicing is controlled by pre-mRNA sequences (cis-elements) and trans-acting protein factors that bind them. The combinatorial interactions of multiple protein factors with the cis-elements surrounding a given alternative splicing event lead to an integrated splicing decision. The mechanism of multifactorial splicing regulation is poorly understood. Using a splicing-sensitive DNA microarray, we assayed 352 Caenorhabditis elegans alternative cassette exons for changes in embryonic splicing patterns between wild-type and 12 different strains carrying mutations in a splicing factor. We identified many alternative splicing events that are regulated by multiple splicing factors. Many splicing factors have the ability to behave as splicing repressors for some alternative cassette exons and as splicing activators for others. Unexpectedly, we found that the ability of a given alternative splicing factor to behave as an enhancer or repressor of a specific splicing event can change during development. Our observations that splicing factors can change their effects on a substrate during development support a model in which combinatorial effects of multiple factors, both constitutive and developmentally regulated ones, contribute to the overall splicing decision.