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The precision and complexity of intron removal during pre-mRNA splicing still amazes even 26 years after the discovery that the coding information of metazoan genes is interrupted by introns Berget et al. Adding to this amazement is the recent realization that most human genes express more than one mRNA by alternative splicing, a process by which functionally diverse protein isoforms can be expressed according to different regulatory programs. Given that the vast majority of human genes contain introns and that most pre-mRNAs undergo alternative splicing, it is not surprising that disruption of normal splicing patterns can cause or modify human disease.

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. The identification stimulated theories for protein diversity, such as alternative splicing, which over time have been realized repeatedly through experiments. Constitutive splicing is the process of intron removal and exon ligation of the majority of the exons in the order in which they appear in a gene.

RNA splicing

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. The identification stimulated theories for protein diversity, such as alternative splicing, which over time have been realized repeatedly through experiments. Constitutive splicing is the process of intron removal and exon ligation of the majority of the exons in the order in which they appear in a gene.

Alternative splicing is a deviation from this preferred sequence where certain exons are skipped resulting in various forms of mature mRNA. Weaker splicing signals at alternative splice sites, shorter exon length or higher sequence conservation surrounding orthologous alternative exons influence the exons that are ultimately included in the mature mRNA 5. This process is mediated by a dynamic and flexible macromolecular machine, the spliceosome, which works in a synergistic and antistatic manner as explained below 6 , 7.

Three possible mechanisms, exon shuffling, exonization of transposable elements and constitutively spliced exons, have been proposed for the origin of alternative splicing 8. Numerous studies have reiterated the critical and fundamental role of alternative splicing across biological systems 9.

The species of higher eukaryotes have been discovered to exhibit a higher proportion of alternatively spliced genes, which is an underlying indication of a prominent role for the mechanism in evolution. Alternative splicing mediates diverse biological processes over the entire life span of organisms, from before birth to death 10 , Conserved splicing to species-specific splice variants play a significant functional role in species differentiation and genome evolution 12 , 13 , as well as in the development of functionally simple to complex tissues with diverse cell types, such as the brain, testis and the immune system.

Alternative splicing even participates in RNA processing itself, from pre- to post-transcriptional events. Thus, alternative splicing has a role in almost every aspect of protein function, including binding between proteins and ligands, nucleic acids or membranes, localization and enzymatic properties.

Taken together, alternative splicing is a central element in gene expression 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. Gilbert W: Why genes in pieces?

Genome sequence of the nematode C. Finishing the euchromatic sequence of the human genome. Zheng CL, Fu XD and Gribskov M: Characteristics and regulatory elements defining constitutive splicing and different modes of alternative splicing in human and mouse.

J Biomol Screen. Trends Genet. Nat Biotechnol. Blencowe BJ: Alternative splicing: new insights from global analyses. BMC Res Notes. Nucleic Acids Res. BMC Genomics. Mol Cell. Plant Sci. View Article : Google Scholar. Trends Biochem Sci. Wang Z and Burge CB: Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA Biol. FEBS Lett.

Chapter 13 RNA splicing

During splicing, introns non-coding regions are removed and exons coding regions are joined together. For nuclear-encoded genes , splicing takes place within the nucleus either during or immediately after transcription. For those eukaryotic genes that contain introns, splicing is usually required in order to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing is carried out in a series of reactions which are catalyzed by the spliceosome , a complex of small nuclear ribonucleoproteins snRNPs. Self-splicing introns , or ribozymes capable of catalyzing their own excision from their parent RNA molecule, also exist. Several methods of RNA splicing occur in nature; the type of splicing depends on the structure of the spliced intron and the catalysts required for splicing to occur. The word intron is derived from the terms intragenic region , [1] and intracistron , [2] that is, a segment of DNA that is located between two exons of a gene.

Mechanism of alternative splicing and its regulation (Review)

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In some genes the protein-coding sections of the DNA "exons" are interrupted by non-coding regions "introns". RNA splicing removes the introns from pre mRNA to produce the final set of instructions for the protein. This editing process is called splicing, which involves removing the introns, leaving only the yellow, protein-coding regions, called exons. These splicing factors act as beacons to guide small nuclear ribo proteins to form a splicing machine, called the spliceosome.

Mechanism of alternative splicing and its regulation (Review)

The splicing reactions have no net gain in the no. Yet, a large number of ATP is consumed, not for the chemistry, but to properly assemble and operate the slicing machinery.

RNA Processing

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In the appropriate cell type and at the correct developmental stage, ribonucleic acid RNA polymerase transcribes an RNA copy of a gene, the primary transcript. However, the primary transcript may contain many more nucleotides than are needed to create the intended protein. In addition, the primary transcript is vulnerable to breakdown by RNA-degrading enzymes. Before the primary transcript can be used to guide protein synthesis, it must be processed into a mature transcript, called messenger RNA mRNA. This is especially true in eukaryotic cells. Processing events include protection of both ends of the transcript and removal of intervening nonprotein-coding regions. The ends of the primary transcript are particularly susceptible to a class of degradative enzymes called exonucleases.

snRNA - small nuclear snoRNA - small nucleolar Participate in the splicing and transfer of hnRNA. rRNA processing/maturation/methylation.

Pre-mRNA splicing and human disease

Constitutive splicing and the basal splicing machinery

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