Cardiovascular disease is highly prevalent throughout the world and is the leading cause of morbidity and mortality in the USA. Atherosclerosis results from the interaction of environmental and genetic risk factors, including age, sedentary life style, cigarette smoking, dyslipidemia, hypertension, obesity, and diabetes mellitus. About 50% of susceptibility to cardiovascular disease is genetic including polymorphisms affecting risk factors . Despite widely prescribed drugs such as statins to lower cholesterol levels, there is wide range of variability in drug response in terms of both lipoprotein and cardiovascular risk reduction. Identifying genes responsible for coronary atherosclerosis and biomarkers for quantifying an individual risk of cardiovascular disease will enable personalized medicine based on the individual's genetic variants.
Several cis-acting SNPs that regulate alternative splicing of HMGCR and LDLR have been identified by genome-wide association analysis (GWAS) to contribute to inter-individual variation in plasma LDL-cholesterol in multiple independent populations . Among them, a common polymorphism (rs688) within low density lipoprotein receptor (LDLR) has been shown to be associated with increased plasma LDL-cholesterol via promoting LDLR exon 12 skipping . In addition, several of the mutations within LDLR associated with FH disrupt normal LDLR pre-mRNA splicing result in reduced LDLR cell surface protein and LDL internalization . A 10-candidate-gene association study of the prospectively executed Pravastatin Inflammation/CRP Evaluation (PRINCE) study have found that two highly linked SNPs (rs17244841 and rs17238540) in the 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) gene (the direct target of statin inhibition) were associated with variable lipid response such that individuals with the two minor alleles had ~20% smaller reduction in total cholesterol and LDL than individuals with two major alleles of the gene . Our lab recently reported that these two SNPs were also associated with reduced simvastatin efficacy in the Cholesterol and Pharmacogenetics (CAP) study and formed a haplotype (H7) with a third SNP (rs3846662) that was associated with variation in the proportion of HMGCR exon 13 that is alternatively spliced . HMGCR transcripts lacking of exon 13 encodes part of the statin-binding domain of the enzyme. Our results indicate that variation in the production of an HMGCR isoform (exon 13 skipped) with reduced statin sensitivity is a marker of inter-individual differences in low-density lipoprotein cholesterol response to statin treatment . Modulation in pre-mRNA splicing by the targeted therapies have been attempted with promising results. For example, a recent report demonstrated that specific knock-down pre-mRNA of ApoB100, a key player in the development of atherosclerosis, with anti-sense oligonucleotides induced the skipping of exon 27, generating a shorter variant that has been associated with reduced LDL-cholesterol levels . These findings suggest that alternative splicing is a physiologically and clinically relevant regulator of cholesterol metabolism and may be a marker or signal for drug efficacy evaluation. From the therapeutic perspective, the process of alternative splicing is a potential therapeutic target as it may influence the development of human disease.
1.2 Alternative RNA splicing
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Alternative splicing is a mechanism for generating multiple mRNA transcripts from a given gene. This process plays an important role in gene regulation and generating proteomic diversity. Recent studies indicate that nearly 95% of human multi-exon genes undergo alternative splicing . About 80% of this alternative splicing falls within open reading frames (ORFs), and the 20% falls within untranslated regions. About 35% of inferred human alternative splicing isoforms in the RefSeq database were predicted to be subjected to nonsense mRNA mediated decay (NMD) . Regulation of alternative splicing controls the expression of functionally diverse isoforms, post-transcriptional regulatory responses and on-off regulation by NMD. Choices of alternative splicing are made at the stages of splice site recognition and different stages of splicesome assembly. In addition, there is considerable evidence for transcription-coupled alternative splicing through RNA polymerase II and transcription factors interacting directly or indirectly with splicing factors .
Pre-mRNA splicing is carried out by the splicesome, a complex macromolecular machine, in which five small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5, U6 and 100 non-snRNP splicing factors cooperate to recognize the splice sites and catalyse the splicing reaction .Splice site choice involves an interplay of cis-acting sequence elements and trans-acting factors. Splicing factors recognize either splicing enhancers or silencers, which are either exonic or intronic, leading to exon inclusion or skipping respectively. Key pre-mRNA splicing regulatory elements are indicated in figure 1.
Antagonism in splice site recognition
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Splicing of pre-mRNA is determined by combinatorial or competitive effects of cis-acting elements and both activator and inhibitors, often by SR proteins and hnRNPs. SR family of proteins contain one or two N-terminal RNA recognition motives (RRMs) and a region of variable length that is enriched in repetitive Arg-Ser dipeptides (RS domain). RS domains mediate protein-protein interaction with other RS-domain-containing splicing factors as well as with the pre-mRNA. SR proteins are involved in various steps of splicesome assembly. SR proteins (specifically ASF/SF2 and/or SC35), bind to ESEs , and help enhancer complex to the 5' and 3' splice site through a network of protein-protein interaction with u1 70K and/or U2AF, thereby facilitating splice site recognition. By contrast, hnRNP family proteins antagonize this activity of SR proteins by blocking binding of snRNPs to regulated exons. For example, hnRNP A1 binds to upstream and downstream of exon 7B in its own pre-mRNA to induce exon 7B skipping . hnRNP A1 and hnRNP I(also known as polypyrimidine tract binding protein 1 PTBP1) have been identified as key splicing repressor. SR proteins and hnRNP proteins involved in pre-mRNA splicing are listed in table 2.
Regulation of alternative splicing by PTBP1
Negative regulation of exon inclusion is a critical layer splice site choice. PTBP1 has been identified as a key splicing repressor in mammalian cells. PTBP1 has also been implicated in regulating other processes such as mRNA stability, mRNA location, polyadenylation and internal ribosome entry site-driven translation . For example, it has been recently reported that PTBP1 modulates LDLR mRNA stability through specific interaction with the Alu-rich elements in the 3'-UTR . These regulation processes are not discussed here. We address evidence that PTBP1 is a key regulator of splicing and its major effect on pre-mRNA is exon silencing.
PTBP1 is composed of four RNA-recognition motif (RRM) domains with interdomain linker regions and an N-terminal extension containing both nuclear localization and export signals. One of the mechanisms for PTBP1-mediated splicing repression is that PTBP1 binds to CU-rich elements flanked by pyrimidines, overlapping with the U2AF65-binding sites near the 3'-splice site, and thus compete with U2AF65 binding. PTBP1 also binds to intronic splicing silencers (ISS) in a long list of alternatively spliced pre-mRNAs resulting in inhibiting intron definition, including c-src, alpha-actinin, FGF-R2, Calcitonin, GABA gama-2, alpha-tropomyosin. Mutation of the PTBP1 binding sites reverses exon silencing in vivo and PTBP1 binding in vitro. Mechanisms underlying above examples involve PTBP1 inhibits intron and/or exon definition or the multimeric binding of PTBP1 to substrate RNAs either by "propagative binding" or by looping of RNA between high-affinity sites, and thus blocking access of splicing factors to their binding sites on the pre-mRNA.
Although PTBP1 is a well-characterized splicing repressor, recent splicing array analyses revealed PTBP1 regulates both exon inclusion and skipping in vivo . Genome-wide analysis of PTBP1-RNA interactions indicate that dominant PTBP1 binding close to the alternative splice site is associated with exon skipping, whereas overriding PTBP1 binding near a competing constitutive splice site is correlated with exon inclusion. These findings suggest a general mechanism for PTBP1 to regulate splice site competition to generate opposite functional consequences. In addition, a recent splicing array study has shown it is common that one alternative splicing regulator is controlled by another splicing regulator and may create a variety of feedback or feed forward regulation at the splicing level. It is important to point out that the final splicing decisions are determined by the sum of competing binding events modulated by multiple different splicing regulators, which may act as synergistically or antagonistically. Therefore, it is possible that other regulators may override the effect of PTBP1 may account for various exceptions other than the positional effect.
Alternative splicing and tissue specificity
The regulation of alternative splicing by tissue-specific factors is well recognized. High-throughput studies have shown that more than 50% of alternative splicing isoforms are differentially expressed among human tissues . Numerous tissue-specific alternative splicing regulators have been identified suggesting that tissue-specific alternative splicing can be explained, at least in part, by tissue-specific expression of splicing factors, and the corresponding regulation of their target transcripts. Among all human tissues, brain has the most diverse tissue-specific alternatively spliced isoforms. Several brain-specific splicing factors have been identified, including nPTB( Neural PTB) , NOVA1, NOVA2 and Hu/Elav proteins. nPTB is structurally and functionally similar to PTBP1 . However, nPTB is only highly expressed in neurons, while PTBP1 is widely expressed in non-neuronal cells. Although PTBP1 is expressed in neural progenitor cells, its expression is down-regulated in differentiated neurons, where nPTB is up-regulated. A recent splicing array coupled with single and double RNAi knock down study revealed that the alternative exons regulated by PTB differ significantly from those regulated by nPTB and double knock down produces a further distinct splicing profile . These findings suggest that PTBP1/nPTB switch provides a post-transcriptional mechanism for programming neuronal differentiation. Tissue-specific alternative splicing factors such as RBM35a and RBM35b have been identified to be important in controlling the expression of epithelial cell-specific exons . Taken together, all these findings suggest that alternative splicing plays an important role in defining tissue specificity.
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Signal pathways affecting alternative splicing
There is an accumulating evidence showing that extracellular signals such as hormonal or metabolic signals, on the regulation of alternative splicing via the activation of diverse signal transduction pathways, which is a relatively new area of research .
A most thoroughly characterized model is alternative splicing of CD44 exon 5. Phorbol esters signals actives the Ras-raf-MEK-ERK pathway and the final target is Sam68, which gets phosphorylated, binds a 10-nucleotide cis-acting element with the composite splice regulator, and inhibits hnRNP A1 in CD44 splice variant CD44 v5 . Apoptosis stimulating protein of p53 (ASPP1) has also been shown to interact with Sam68 and regulate CD44 v5 alternative splicing in vivo . Other stimuli have been shown to alternative splicing include dexamethasone, insulin, cytokine, Src, UPR, calcium, TPA, PDGF, IGF-1, NGF and serum .
Very little is known about the nuclear control of alternative splicing switch after hormonal or metabolic signals. The general approach is to identify components of the nuclear splicing machinery and to test the effects of signals on these components. The assembly of the splicesome complex involves phosphortlation and/or dephosphoryltaion of splicing factors . There are some data suggesting that splicing factors may be regulated by hormonal signals. For example, insulin treatment of a H35 cells, which is a rat liver cells, increased phosphorylation of SRp75 and induced fibronectin gene alternative splicing. Signalling pathway may also influence the localization of splicing factors. The shuttling of the hnRNP A1 between nucleus and cytoplasma is regulated via the MKK3/6-p38 pathway, leading to an altered ratio of hnRNP A1 to its antagonist SR protein SF2/ASF in the nucleus and thus decreasing E1A 9S isoform relative levels.
The examples described above shed light on the role of alternative splicing regulation as a fine and specific tuning of the cellular response to different stimuli or different physiological , pathological conditions.
Alternative splicing resulting in nonsense-mediated mRNA decay
Alternative splicing can introduce premature termination codons (PTCs). Numerous studies have shown that PTC-containing mRNAs are expected to be degraded by nonsense-mediated mRNA decay (NMD). A stop codon is recognized as PTC if it is located >50-55 nucleotides (nt) upstream of an exon-exon junction in mammals. By using computational approaches and microarry platforms, Brenner and colleagues found that ~35% of inferred human alternative splicing isoforms in the RefSeq database were predicted that to be subjected to NMD . NMD not only degrades transcripts that are the consequence of routine abnormalities in gene expression but also is widely used to achieve proper levels of gene expression . Interestingly, alternative splicing coupled with NMD has been found to occur within individual members of splicing factors-both the SR and hnRNP family proteins . These results were confirmed by Saltzman et al.
1.4 LDLR function and regulation
The low density lipoprotein receptor (LDLR) is a cell surface transmembrane protein that mediates the uptake of plasma LDL particles and regulates cholesterol homeostasis in mammalian cells. Mutations in the LDLR gene cause the autosomal dominant disease familial hypercholesterolemia(FH), which promotes premature coronary atherosclerosis.
The LDLR gene has been mapped to chromosome 19p13.2 and is approximately 45 kb in length. The LDLR protein is a multi-domain protein whose extracellular domain is composed of an N-terminal ligand binding domain that mediate binding to LDL and Î²-VLDL, followed by the epidermal growth factor(EGF)-precursor homology domain (with the EGF-like repeats A, B and C, as well as a Î²-propeller between B and C) . Ligands bound extracellular domain of the LDLR are internalized and then released in the endosomes, leading to their lysosomal degradation.
The LDLR gene is regulated by the sterol-responsive element binding protein-2 (SREBP-2). In cholesterol -depleted cells, SREBP-2s are transported to Golgi, where they are cleaved in tandem by the pro-protein convertase SKI-1/S1P and the intramembranous metalloprotease S2P to release a soluble fragment to enter the nucleus and up-regulate transcription of the LDLR and other genes in the cholesterol pathway. In LDL-derived cholesterol-loaded cells, SREBP2s are blocked by the LDL-cholesterol, thereby blocking the proteolytic release of the active fragment SREBP-2s. Transcription of the LDLR declines thus preventing cholesterol overload. Proprotein convertase subtilisin/kexin type 9 (PCSK9) enhances the degradation of the LDLR protein. Both LDLR and PCSK9 are positively regulated by the SREBP-2. Sterol depletion causes two opposing effects on LDLR protein.
1.3 Alternative splicing of the LDLR
LDLR undergoes alternative splicing to produce several different transcripts, of which those lacking exon 4 or exon 12 are the most common. Exon 4 skipping maintains the open reading frame, while exon 12 skipping interrupts the open reading frame and introduces a premature stop codon. Both exon 4 and exon12 skipped transcripts have been shown to reduce LDLR cell surface protein and LDL internalization . A common polymorphism within LDLR, rs688, has been shown to be associated with increased plasma LDL-cholesterol levels and to promote exon 12 skipping . In addition, several of the mutations within LDLR have been shown to disrupt normal pre-mRNA splicing, resulting in reduced LDLR cell surface protein and LDL internalization .
LDLR exon 4 skipping maintains open reading frame, while exon 12 skipping disrupts the reading frame and introduces a premature termination codon. It remains unknown whether LDLR transcripts lacking of exon 12 undergo NMD.
1.7 Model system for investigating regulation of alternative splicing
Regulation of alternative splicing of LDLR in human can not be determined directly in vivo for ethical and technical reasons. Although the high degree of sequences and structural conservation are observed in both the LDLR gene and protein of human, rat, and hamster, there are substantial cross-species differences in alternative splicing patterns of the LDLR gene. None of the LDLR mRNA lacking exon 4 or exon 12 was detected among mouse and hamster EST sequences by a computational prediction of alternatively spliced isoforms based on mouse EST sequences using the Extended Alternatively Spliced EST Database and Blast , indicating that the LDLR alternative splicing of exon 4 or 12 are not conserved. Therefore, we are limited to experimentation in human derived cells, such as human hepatocyte HepG2 cells, fresh or immortalized lymphocyte cell lines, human fibroblasts.
Most of LDLR deficiency was obtained with human familial hypercholesterolemia (FH) fibroblasts; however, analysis of the importance of the hepatic LDLR is more relevant as the liver is a major site of clearance of plasma LDL cholesterol through LDLR in mammals. It has been demonstrated both sterol-dependent and sterol-independent LDLR regulation in human hepatoma HepG2 cells has been used for elucidating molecular mechanisms of cholesterol homeostasis by many studies .
It has been demonstrated that both freshly isolated and transformed lymphocytes can be used for studying genetically regulated variation in cholesterol metabolism . In addition, our lab has shown that cholesterol homeostasis is faithfully maintained in immortalized vs. freshly isolated lymphocytes. When repositories of immortalized lymphocyte cell lines are derived from clinical trials, in vitro cellular phenotypes can be correlated with phenotypes measured in vivo in the donor individuals.
1.8 Alternative splicing of genes involved in cholesterol metabolism
It has been reported that ~90% of human genes undergo alternative splicing . Alternative splicing has been reported in other genes besides LDLR involved in cellular cholesterol biosynthesis and LDLR degradation. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthesis pathway, undergoes functionally important alternative splicing of exon 12, resulting in a second transcript, HMGCR 13(-). Our lab recently has found that inter-individual variation in HMGCR alternative splicing is likely a determinant of LDL-cholesterol response to simvastatin , suggesting that alternative splicing is a physiologically and clinically relevant regulator of cholesterol metabolism. 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1 soluble), catalysing the first step cholesterol biosynthesis pathway, has an optional exon 2 in the 5'-UTR region, which may regulate translational efficiency. Mevalonate kinase (MVK), converting mevalonate into 5-phosphomevalonate in the step following HMGCR, has been found to undergo alternative splicing of exon 4 and/or exon 5. Both disrupt the open reading frame and are predicted to be degraded by NMD. An in-frame splice variant of PCSK9 (exon 8 skipped) has been identified and the resulting protein is not functionally active. Alternative splicing of genes involved in cholesterol metabolism have been shown to attenuate or abolish enzyme or protein activity suggests that alternative splicing may be a general regulatory mechanism in cholesterol metabolism, similar to the coordinated transcription regulation mediated by SREBP-2.
LDL particles typically carry a majority of the circulating cholesterol and it has been clearly shown that these particles are atherogenic.
Alternations in splicing can cause disease directly, link to disease susceptibility, drug efficacy and toxicity.