The remaining 1. The size of genes varies, with an average estimate of 30, base pairs bp per gene in humans. Many genes are larger than , bps, with the largest known gene dystrophin being approximately 2.
However, the average size of mature mRNA that codes a protein is usually shorter than 2, nucleotides 8. More interestingly, it has been determined that many individual exons or introns may be included or excluded in some mRNAs, but not in other mRNAs, through an alternative splicing AS process, leading to generation of multiple protein isoforms from a single gene.
Extensive studies have demonstrated that splicing and alternative splicing regulate almost every biological process, including signal transduction and energy transfer in metazoan and plants. A variety of diseases in humans have been found to be caused by defects in pre-mRNA splicing 5 , Correction of defective splicing has recently become a target for the treatment of such diseases in humans.
However, in contrast to humans and other vertebrates, studies of RNA splicing and alternative splicing in plants, such as in rice, are limited. But the mechanisms of splicing in both metazoans and plants are believed to be similar, although many differences are known to exist in actual splicing between metazoans and plants, as discussed below.
Pre-mRNA splicing is a two-step process that involves the formation of phosphodiester bonds The splicing process is not complete until the cleaved exons are ligated together, to form a mature RNA. RNA splicing is a heavily regulated biological process that is dependent on sequence elements in pre-mRNAs. In contrast to metazoans, the branch point and polypyrimidine tract are less conserved in plants. The overall coordination among splicing signals, enhancers, and inhibitors, as well as other components that are discussed below, leads to the precise and orchestrated event of pre-mRNA splicing and alternative splicing.
The spliceosome is a dynamic structure that undergoes multiple complex transitions during the splicing process. Subsequently, a pre-spliceosome complex is formed. Subsequently, it was determined that RNA splicing is a universal event that occurs in all organisms. However, the type and mechanism of splicing varies among species. On the other hand, in eukaryotes, splicing is mostly referred to as trimming introns and the ligation of exons in protein-coding RNAs.
Another major difference in splicing between prokaryotes and eukaryotes is that splicing in prokaryotes does not involve a spliceosome. The frequency of RNA splicing depends on the complexity of gene structures of genomes in particular species.
Generally, many more RNA splicing events occur in higher species, such as mammals, compared to lower species, such as single cell organisms like Saccharomyces cerevisiae yeast Table 1.
As such, splicing is not necessary in these genes. In fact, genes in humans have an average of exons Therefore, most genes in humans undergo splicing, to generate mature mRNA. The human genome contains approximately 25,, genes, a number that is much smaller than initially estimated. One explanation for the smaller number of genes in humans, is that many genes undergo alternative splicing AS , leading to multiple protein isoforms from a single gene, which in turn results in a much greater number of proteins, compared to genes.
Alternative splicing is an event that is found in both metazoans and plants. Since its discovery in early , during characterization of the immunoglobulin mu and calcitonin gene 24 , 25 , alternative splicing has been described in many genes of different species. Alternative splicing plays an important role in the regulation of gene expression, by affecting mRNA stability, through nonsense-mediated decay NMD and translation efficiency Alternative splicing events are heavily regulated at different developmental stages, in different tissues, in different cell types and under different conditions.
Abnormal alternative splicing has been implicated in a number of human diseases, such as cancers breast and lung cancers via the Bcl-x gene 26 , neurodegenerative diseases spinal muscular atrophy via SMN splicing 27 , frontotemporal dementia with parkinsonism FTFP via tau splicing 28 , and other diseases Strategies have been developed to target abnormal alternative splicing in these diseases, as potential treatments 5 , In fact, several clinical trials are underway, to evaluate the potential to treat diseases by correcting aberrant splicing in humans.
Similar to humans, the majority of genes in rice contain multiple exons and introns, therefore requiring splicing to generate mature mRNAs. Although mechanisms of splicing through the spliceosome have been well characterized in humans, as described above, spliceosomes have yet to be isolated from rice, or other plants.
Nonetheless, it is assumed that rice has identical splicing machineries, as in humans. But unlike in humans, studies of alternative splicing in rice have not been as extensive, and most studies have mainly focused on individual genes or gene families.
Subsequently, alternative splicing has been found in many other genes in plants, from such species as Arabidopsis and rice. Most of such genes are involved in splicing, transcription, flowering regulation, disease resistance, enzyme activity and other biological processes, in response to conditions such as stress and salinization Although alternative splicing has been described in many of these genes in different plant species, it was believed that AS in plants was not common.
With growing collections of ESTs, and more extensive coverage of plant genomes, researchers have begun to reevaluate splicing and alternative splicing in rice and other plants. In addition, recent advancements of microarray technologies and next generation sequencing have provided extra tools and greater opportunities to examine the depth of splicing and alternative splicing in rice 30 , 37 - As a result, numerous papers published during the last few years have demonstrated extensive alternative splicing events in rice.
In one of such papers, Zhang et al. A higher percentage of genes that undergo alternative spicing in rice has been reported by other groups While These results suggest that, similar to humans, AS also plays an important role in gene regulation, protein diversity and complexity in rice.
A majority of the 25, genes in humans and the 50, genes in rice contain multiple exons and introns. Therefore, to produce mature mRNAs, splicing is required in both humans and rice.
Humans and rice share a number of similarities with regard to the regulation of splicing and alternative splicing, which are controlled by splicing signals, cis-elements and trans-factors.
These two sites guarantee precise cleavage between introns and exons. In addition, similar to humans, rice contains many homologues of Uridylate-Rich Small Nuclear RNAs UsnRNAs and protein components of spliceosomes, indicating that splicing in rice likely occurs via a spliceosome complex. However, several major differences with regard to the regulation of splicing also exist between humans and rice. First, human genes typically have massive size variations in introns and exons, with an average intron of bps, and small internal exons bps 8 , Second, a branch point is usually required for the splicing of pre-mRNA in humans.
Instead of a polypyrimidine tract, a U-rich sequence in rice pre-mRNA was discovered, in place of a polypyrimidine tract 38 - Due to these similarities and differences in splicing signals, regulatory elements and gene structures, one would expect similarities and differences in splicing patterns between humans and rice.
While constitutive splicing in both humans and rice complies equally well with splicing rules via spliceosome machineries, differences in the types of alternative splicing and extent of alternative splicing are also noticeable. One explanation for this difference is that the coverage of rice transcriptome and collections of ESTs in rice are not yet as extensive as in humans. However, more exons and introns in an average human gene Table 1 may also contribute to the difference, that humans have a higher percentage of genes with alternative splicing.
Seven types of alternative splicing have been observed in both humans and rice Fig. Exon skipping occurs when a middle exon is skipped, leading to two separate exons joining together. Interestingly, it has been previously reported that the average size of retained intron is only bp, which is much smaller than the average size of introns bp in rice, suggesting that intron-retention alternative splicing is related to the size of introns.
Such results are consistent with the hypothesis that organisms such as rice with average small introns, use intron-definition splicing mechanisms, by which introns are initially recognized by spliceosome; whereas organisms like humans with large introns, use exon-definition splicing mechanisms, by which exons are first recognized by splieosomes. One other aspect of alternative splicing between humans and rice that needs to be discussed is cis regulatory elements. These elements are primarily targeted by splicing trans-factors for positive SR proteins or negative regulation hnRNPs: heterogeneous nuclear ribonucleoproteins of alternative splicing of specific genes.
Finally, it should be noted that Zhang et al. Trans-splicing is coined as a splicing event in which chimeric RNAs comprise exons from two or more different genes 38 , Although this is thought to be a rare occurrence, the number of trans-splicings may be higher than expected. Identification and characterization of trans-splicing RNAs may provide insight on how genes are regulated in both humans and rice.
Regulation of splicing and alternative splicing is an exquisite biological process that requires not only splicing signals, cis-elements and gene structures, but also many protein and RNA components, to form spliceosoms. In addition, trans-elements such as SR proteins and hnRNPs are essential for the regulation of alternative splicing in different organs, at different developmental stages, and under different conditions. Among trans splicing elements, SR proteins are the most well studied group of splicing factors 16 , SR proteins play key roles in regulating both the constitutive and alternative splicing of many genes, including their own genes, in all species 29 , 44 - However, until as recently as , it was ambiguous as to which proteins actually constituted member of the SR protein family, because proteins with RS domains but no RRP motifs exist.
To simplify classification, Manley and Krainer 46 redefined SR proteins, based on studies in mammals, as proteins with one or two N-terminal RRMs, followed with a downstream c-terminal arginine serine-rich domain RS domain Table 2. However, this definition would have excluded a number of proteins in plants from SR families.
Therefore it was later clarified by Barta et al. In addition, it was found that another rice specific subgroup has two RRMs. Similar to this classification, in a more recent study Richardson et al. They classified SR proteins into five major groups, which can be further divided into 11 sub-groups. Five of these sub-groups are mainly composed of plant SR proteins, while the six remaining subgroups are mainly comprised of mammal SR proteins.
Systematic analyses of ESTs and microarray data have so far revealed seven main types of alternative splicing 12 Fig. Intron retention in human transcripts is positioned primarily in the untranslated regions UTRs 16 and has been associated with weaker splice sites, short intron length and the regulation of cis-regulatory elements Five main types of alternative splicing events are depicted.
One example of a transcript that undergoes alternative splicing, which generates variation in the protein, is FGFR2. Of note, it has been demonstrated that each type of alternative splicing can operate in a stochastic manner, and different splice-site identification and processing mechanisms do not necessarily occur at the same frequencies among all biological kingdoms The mechanisms outlined above are just one indication of the complexity, as numerous molecules are involved in alternative splicing in a coordinated manner.
Even the basic nucleotide components and the essential molecules that recognize them can introduce diversity in the synthesis of mature transcripts. Two major steps constitute the basic process of splicing: Assembly of the spliceosome followed by the actual splicing of pre-mRNA. Numerous steps in the pathway are reversible The diagram illustrates the appropriate relative distributions of the molecules and core splicing signals with its consensus sequence in regulation of the alternative splicing.
The enhancer elements [ exonic splicing enhancers ESEs and intronic splicing enhancers ISEs ] are recognized by activator proteins the SR protein family , and the silencer elements [exonic splicing silencers ESSs and intronic splicing silencers ISSs ] are bound by repressor proteins [the heterogeneous nuclear ribonucleoproteins hnRNP protein family]. These two protein families are engaged to promote or inhibit spliceosome assembly at weak splice sites, respectively.
The exons that end up in the mature mRNA during the process of alternative splicing is entirely defined by the interaction between cis-acting elements and trans-acting factors. The collaboration between these elements results in the promotion or inhibition of splicesome assembly of the weak splice sites, respectively Fig.
In general, the cis-acting elements function additively. The enhancing elements tend to play dominant roles in constitutive splicing, while the silencers are relatively more important in the control of alternative splicing Enhancer activity has been shown to be abolished by a stable stem-loop structure as short as 7 base pairs in an RNA transcript owing to the mechanisms of physical competition, long-range RNA pairing, a structure splice code and co-transcription splicing 24 , Furthermore, the specificity of cis-acting enhancer elements for introns or exons has been investigated.
Similarly, the antagonistic role of hnRNP M to the splicing factor Nova-1 generates alternatively spliced dopamine receptor pre-mRNAs, which create isoforms associated with diverse key physical functions, such as control, reward, learning and memory In general, positive or negative splice-site recognition is regulated through various mechanisms, such as the local concentration or activity of splicing regulatory factors, under diverse physiological or pathological conditions.
How these elements function together to precisely select a regulated splice site is, however, only partially explained by these results Since the first significant observation of co-transcriptional spliceosome assembly from electron micrographs of Drosophila melanogaster embryonic transcription units 37 , increasing evidence supports the idea that transcription and splicing are physically and functionally coupled, and has also uncovered the intricate association between mRNA splicing, RNA polymerase II Pol II and chromatin structure 38 , A large number of components associated with the physical interaction between splicing and transcription have been purified, with particular attention on the carboxyl terminal domain CTD of the large subunit of RNAPII The CTD consists of 52 tandem repeats of the heptapeptide YSPTSPS in mammals 26 tandem repeats in yeast 41 , which act as a special platform to recruit different factors to the nascent transcripts via dynamic phosphorylation of serine residues.
Kinases that phosphorylate specific CTD serine residues have been identified and are components of the protein apparatus driving the specific function. In addition, phosphorylation of ser7 has been found to facilitate elongation and splicing Thus, phosphorylation is a mechanism that clearly demonstrates that functional coupling exists between transcription and alternative splicing. Of note, mutation and deletion analysis of CTD has revealed multiple defects in mRNA processing 45 , therefore, CTD and additional components of the two machineries have emerged as a central element in governing the interactions between transcription and splicing.
Taken together, functional coupling appears to maintain an important role in alternative splicing in driving determinative physiological changes, and fine-tune gene expression in mathematical modeling approaches Two models have been suggested to explain the co-transcription process of how transcription coupled repair influences alternative splicing.
The mechanism of the recruitment model may mainly depend on specific features of CTD as mentioned above , whereas the kinetic model is based on the different elongation rates of Pol II, which in turn determine the timing of the presentation of splices sites 47 , Fundamentally, the aforementioned mechanism influences patterns of alternative splicing via the variations in Pol II elongation and recruitment of splicing factors by specific histone marks Thus, alternative splicing is highly influenced not only by transcription, but also by the chromatin structure, which underscores chromatin as another layer in the regulation of alternative splicing.
The resultant mature mRNA is thus a reflection of numerous DNA modifications, such as patterns of histone methylation at exons, modulation of histone modifications and increased DNA methylation at exons 50 , Conversely, a previous study indicated that splicing may mediate chromatin remodeling via deposition of histone marks on DNA or numerous associations between splicing factors and elongation proteins Adding additional complexity to the regulation network is alternative transcription initiation ATI and alternative transcription termination ATT sites.
ATI and ATT significantly contribute to the diversity of the human and mouse transcriptomes to a degree that may exceed alternative splicing, when considering the number of possibilities available through alternative nucleotides, isoforms and introns 52 , By contrast, alternative splicing associated alterations mostly lie within the protein sequence, potentially affecting almost all areas of protein function 14 , NMD is an extensive and complicated mechanism, ranging from yeast to human, exploited to achieve another level of robustness in post-transcriptional gene expression control.
Furthermore, analysis of quantitative alternative splicing microarray profiling has demonstrated that individual knockdown of NMD factors [Up-Frameshift UPF ] strongly affects PTC-introducing alternative splicing events, indicating a role for different UPF factor requirements in alternative splicing regulation In a second example, regulation of intron retention by alternative splicing-NMD in a specific differentiation event has been recently observed Trans-splicing is a common phenomenon in trypanosomes, nematodes, Drosophila and even humans, and refers to the novel and unusual splicing of exons from independent pre-mRNAs 62 , The phenomenon has been explored as a therapeutic option for a variety of genetic diseases, particularly in the treatment of cancer The carcinoembryonic antigen CEA , for example, is associated with a variety of neoplastic processes and was exploited as a target for trans-splicing.
The activity of the ribozyme simultaneously reduced CEA expression and introduced the thymidine kinase gene, which rendered the cells sensitive to ganciclovir treatment. RNA trans-splicing has also been utilized for the potential treatment of neurodegenerative diseases through a novel technology, spliceosome mediated trans-splicing SMaRT. SMaRT was successfully used in vivo to re-engineer tau mRNA transcripts to include E10, and therefore, offers the opportunity potential to correct tau mis-splicing and treat the underlying disease Non-coding RNAs ncRNAs , including microRNA and small interfering RNA, have recently emerged as novel regulators in alternative splicing, generally through the modulation of the expression of key splicing factors during development and differentiation Stringent regulation of alternative splicing is necessary for the functional requirements of complex tissues under normal conditions, whereas aberrant splicing appears to an underlying cause for an extremely high fraction of dysfunction and disease Aberrant splicing has been suggested to root in alterations of the cellular concentration, composition, localization and activity of regulatory splicing factors, as well as mutations in components of core splicing machinery A changed efficiency of splice site recognition is the immediate consequence, while irregularities in protein isoforms in different systems ultimately establish the disease state.
Any of these alterations affecting alternative splicing can facilitate the appearance of characteristics in cancer cells, including the inappropriate proliferation, migration, methylation changes and resistance to apoptosis and chemotherapy Alternative splicing has been implicated in nearly all aspects of cancer development, and therefore, is a main participant in the disease. Understanding the basic mechanisms and patterns of splicing in tumor progress will shed light on the biology of cancer and lay the foundation for diagnostic, prognostic and therapeutic tools with minimum treatment toxicity in cancer Extensive research efforts have already committed to developing drugs that target specific cancer protein isoforms.
However, limited success has been achieved by simply activating or inhibiting cancer-associated genes, possibly due to the expression of target genes in normal and cancers cells, such as angiogenic and anti-angiogenic isoforms The lack of specificity of numerous molecular targets for cancer cells favors the development of isoform-specific diagnostic markers as therapeutic targets Therefore, the key task for cancer treatment in the future should be to detect and target the expression of a gene at the gene level.
The combination of an alternative splicing database, tandem mass spectrometry, and even the latest synthetic alternative splicing database may aid with the identification, analysis and characterization of potential alternative splicing isoforms.
Alternative splicing appears to be prevalent in almost all multi-exon genes. All these deficiencies lead to an incomplete understanding of the alternative splicing mechanism and may prevent the correct prediction of splice events in other species, such as the chimpanzee or plant 77 , Distinguishing alternative splicing from other regulatory mechanisms in the gene regulation is also difficult.
Alternative splicing, alternative trans-splicing, NMD, transcriptional efficiency, exon duplication and RNA editing 79 all contribute to an extensive mechanism for generating protein diversity. In addition, the difference between artificial experimental systems and real-life scenarios makes it challenging to transfer functional studies from cells to whole organisms.
Numerous questions remain regarding the global impact of alternative splicing on cellular and organismal homeostasis, as well as its underlying molecular mechanisms. Finally, with regards to cancer-associated alternative splicing, whether a particular splice site selection causes the observed effect or is merely the result of the cancerous transformation is hard to distinguish.
The data collected regarding alternative splicing is likely to represent only the tip of the iceberg, with further information yet to be revealed in future studies. National Center for Biotechnology Information , U. Journal List Biomed Rep v. Biomed Rep. Published online Dec Author information Article notes Copyright and License information Disclaimer. China E-mail: nc. Received Nov 12; Accepted Dec This article has been cited by other articles in PMC. Abstract Alternative splicing of precursor mRNA is an essential mechanism to increase the complexity of gene expression, and it plays an important role in cellular differentiation and organism development.
Keywords: alternative splicing, regulation, precursor mRNA, mechanism, disease. Introduction The discovery of the phenomenon that viral sequences are removed from a pre-mRNA and the remaining sequences are joined together led to a fundamental principle governing biology, known as RNA splicing.
Molecular mechanisms of alternative spicing Systematic analyses of ESTs and microarray data have so far revealed seven main types of alternative splicing 12 Fig. The main difference between RNA splicing and alternative splicing is that the RNA splicing is the process of splicing the exons of the primary transcript of mRNA whereas the alternative splicing is the process of producing differential combinations of exons of the same gene.
Abdelaali Bargas Pundit. How are introns removed? Introns are removed by RNA processing in which the intron is looped out and cut away from the exons by snRNPs, and the exons are spliced together to produce the translatable mRNA. The resulting mature mRNA may then exit the nucleus and be translated in the cytoplasm.
Nixon Madrugo Pundit. Why are introns needed? Eukaryotes might need this diversity in proteins because they have many types of cells all with the same set of genes. Therefore, introns are a way to generate different proteins or different amounts of proteins that are unique to a cell type.
Introns might also allow for faster evolution. Antimo Haidler Teacher. What determines alternative splicing? The discovery of alternative splicing. Jemal Gericke Teacher. Can one gene make different proteins? One gene , many proteins — alternative splicing. This process is called alternative splicing and it makes it possible to produce different proteins from the same gene these different protein versions from the same gene are called isoforms. Beckie Greening Teacher. What is the purpose of alternative splicing in eukaryotic cells quizlet?
In alternative splicing , different combinations of exons from the same gene are combined to result in different protein products. Often, these different splice variants are expressed in different tissues.
This allows for an increased diversity of proteins to be produced. Mariona Blankenstein Teacher. What happens during RNA splicing? RNA splicing is a process that removes the intervening, non-coding sequences of genes introns from pre-mRNA and joins the protein-coding sequences exons together in order to enable translation of mRNA into a protein.
Tayri Peix Reviewer. What is Polycistronic mRNA? At least 15 different mRNAs are produced from this gene cluster. Zakarias Dickhaus Reviewer.
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