# RNA Splicing

RNA splicing is a modification of an RNA that takes place during the transcription of the primary transcript to the mRNA. Splicing refers to introns being cut out or removed, and the remaining sequence (called exons) being attached. This modification occurs in the nucleus, before the RNA is moved to the cytoplasm.
Splicing happens in all the domains of life, but types of splicing differ immensely between the major divisions. Eukaryotes splice many protein-coding messenger RNAs and some non-coding RNAs. Prokaryotes, on the other hand, splice rarely, and when they do, it is mostly non-coding RNAs.

## Discovery of RNA Splicing

RNA splicing was discovered by two scientists Phillp Allen Sharp and Richard J. Roberts and they were awarded the 1993 Nobel Prize in Physiology or Medicine for their achievement. The initial discovery of RNA splicing led to the resolution of an earlier paradox in which scientists had discovered RNA in the nucleus that was unusually long compared to the mRNA found in the cytoplasm of the cell. The strange nuclear RNA had a 5’ end containing a cap structure and a 3’ end that contained a polyadenosine [poly(A)] tract and these were similar structures found in the shorter mRNA found in the cytoplasm. The subsequent discovery of splicing explained how the small mRNA could have the same termini as the longer nuclear RNA. While the termini were the same, the lengths were different because introns had been removed from the middle of the strand. These introns, it was discovered, proved to be a problem for the cell because, for example, a nearly a quarter of all mutations in globin genes responsible for beta-thalassemia came from problems in splicing.
It became apparent through development of reactions that replicated RNA splicing that the splicing is done by a branch-shaped section of a lariat RNA and that such RNAs were integral to splicing. Later it was found that these small snRNAs compiled particles found in spliceosomes. Via an intermediatemade up of lariatRNA and the 5’ exon-RNA, the spliceosome was able to remove the intron.

## Spliceosome

In eukaryotes, genes are transcribed to messenger RNAs comprising both introns and exons. For the production of validated mRNAs, the introns are to be trimmed off and the exons attached back together by the spliceosome, the molecular tailor of the cell. The spliceosome has the ability to alter its snipping and stitching process in order to generate variation in mRNAs based on a single coil of pre-mRNA cloth. Alternative splicing is the process by which the spliceosome can develop multiple mRNA isoforms from a single bolt of pre-mRNA. Such alternative splicing has enhanced evolutionary possibilities in complex multicellular organisms without the addition of gene number.
The spliceosome is considered as one of the most complicated macromolecular machine in the eukaryotic cell. It is involved with hundreds of RNA and protein mechanisms, specifically with assembly and disassembling pathways. The main role for a spliceosome in eukaryotes is to develop messenger RNAs. Genes are transcribed as precursors to mRNAs, called pre-mRNA, and then the RNAs are generated by the snipping and stitching of intron and exon components. Introns are regions of the pre-mRNA that are cut by spliceosomes to serve as a source of non-coding RNAs. Exons code for proteins so they are usually wanted. Furthermore, a spliceosome can uniquely snip and stitch in ways that will create different types of mRNA, which as allowed evolution to allow organisms to increase in gene number and complexity. The reason why Spliceosome are considered one of the most complicated macromolecule machines in a cell is because they have the responsibility of properly recognizing and processing a large amount of sequences. For example, spliceosomes end up processing five small RNAs and up to 100 different polypeptides in budding yeast. To make things more complicated, humans even need to use a second splicing apparatus, the minor spliceosome. In studying these complex machinery, many barriers were in the way because of the limitations in vivo. However, novel approaches to researching splicing have developed, such as in vitro assembly and purification of active spliceosomes, microscopic visualizations of single spliceosomes, and more. The advantages of these methods are that they are more specific and allow the wider boundary of studying either hundreds or a single RNA molecule. One of the new methods involves using microarrays.
Using splicing-dependent microarrays allows researches to distinguish which features of the splicing cycle are universal or specific to pre-mRNAs. Groups of developed DNA arrays differentiate between spliced and unspliced RNAs and then are probed with cellular RNA to isolate even further. Analysis of the splicing response allowed the observation of how loss of activity in specific protein directly affected the splicing of individual pre-mRNA. Overall, the microarray has proved its importance of pre-mRNA identity by efficiently isolating the desired-protein.
Spliceosome analysis have often times brought up significant obstacles in gaining understanding of more detailed mechanisms. To overcome such difficulties, laboratory techniques have employed methods like in vitro, which involves observing single pre-mRNA molecules, and active spliceosome purification, which includes well- characterized enzymes and controlled conditions. Although each chemical approach are relatively distinct, each contribute a complementary and synergistic view that heighten the knowledge of splicing machinery.
The spliceosome is a complex macromolecular machine consisting of small nuclear riobonucleoprotein particles (snRNPs): U1, U2, U4, U5, and U6, as well as roughly 100 separate splicing factors. The snRNAs range in length from 107-210 nucleotides; the snRNAs link with proteins to make small ribonucleoprotein particles (snRNPs). The snRNP contains a single snRNA and multiple proteins.
Splicing is carried out in multi-megadalton complexes. This means that the spliceosome is made of several components in an ordered manner. First, U1 snRNP binds to the 5’ splice site (SS). At the same time, branchpoint bridgeing protein (BBP) and Mud 2 binds to the branch site. Then, U2 snRNP will displace the BBP/Mud 2 and bind to the branch site. Next, U4/U6.U5 tri-snRNP will also binds to the complex. Before splicing the RNA U1 and U4 will leave the complex and Prp19 will bind. After the splicing is completed, the spliceosome will undergo conformational change for the ligation process. After the ligation process, the components of the Spliceosome will degrade and be recycled. Therefore, each spliceosome is a single turnover enzyme
From all the new and developed techniques, it is not for certain that the spliceosome cycle in the body is far from simple, but rather an "extraordinary dynamic and flexible machine" The new technologies constantly bring new evidence of the detailed reversible, irreversible, kinetic, and mechanism interactions of the pre-mRNA substrate. There are still many things unknown about the spliceosome and its process. There is still limited structural information, which means many of its functional details are unavailable. Same with unknown kinetic understanding. However, the research of this dynamic machinery is still developing and continuing to discover new methods and information on its purpose.

## Pre-mRNA Splicing Process

Splicing requires there to be three sequences in the introns. One end of the intron is the 5' splice site and the other end is the 3' splice site. At these sites are short consensus sequences. The sequence that exons are ordered in the mRNA usually correlates with the sequence in the corresponding DNA. The process is aided by spliceosomes, which are small RNA molecules that recognize the beginning of introns (usually GU) and the end (usually AG) and catalyze splicing at these sites. Changing a single nucleotide at these sites may prevent splicing to occur. There are also self-splicing introns. The third sequence important to splicing is located at the branch point. The branch point is where an adenine nucleotide lies from 18 to 40 nucleotides before the 3' splice site. The deletion or mutation of the adenine nucleotide at the branch point would prevent splicing. Splicing occurs in large structures called spliceosomes.
Before splicing takes place, an intron between exon 1 and exon 2. Pre-mRNA splices in two steps. In step one, the pre-mRNA is spliced at the 5' splice site, separating exon 1 from the intron. The 5' end of the intron then attaches to the branch point folding back on itself and forming a structure called a lariat. The folding back occurs by the guanine nucleotide in the 5' consensus sequence bonding with the adenine nucleotide at the branch point through transesterification. In step two a splice is made at the 3' splice site and the 3' end of exon 1 is attached to the 5' end of exon 2. The intron is separated as a lariat and becomes linear when the bond breaks at the branch point and is then degraded by nuclear enzymes. And finally, the mature mRNA consisting of only the exons spliced together are moved to the cytoplasm and translated.
It is important to note that the 5' cap greatly affects pre-mRNA processing and mRNA export and if it were ever to be removed, then it would be known as the first irreversible step in mRNA decay which will affect the entire gene expression.

## Pre-mRNA Splicing: Constitutive vs Alternative

Constitutive splicing pertains to the way mRNA is spliced in exactly the same way, every time with the splicesome
Alternative splicing allows for different expression of genes through SR proteins, which select alternative sites for splicing, using different exons or expressing them in a different order. By choosing combinations of alternative splice sites, protein isoforms can be created that are structurally and functionally distinct. It is estimated that at least 75% of human genes undergo this mechanism.
Another alternative splicing uses multiple 3' cleavage sites. There are 2 or more potential site for cleavage in a pre-mRNA sequence. However, this may or may not produce different proteins.

## Alternative splicing can occur under cellular Stress

Because alternative splicing can control gene expression, it is an important mechanism that a cell can use in response to certain stressful environmental and pathological cellular conditions such as heat, cold, UV-light, oxygen, ion balance, infections, inflammation, fever, etc.
Splicing factors can be enhancing (recognizing positive sequence elements) or silencing (recognizing negative sequence elements). These sequence elements can by exonic or intronic which determines whether they are included (exonic) or left out (intronic). The splicing enhancers are typically bound and activated by SR proteins. SR proteins are ‘serine/arginine-rich;’ they are a group of proteins that have been highly conserved throughout evolution that participate in both alternative and constitutive splicing. They are involved in regulating and selecting the splice sites.
Examples:
Alternative or unconventional mRNA splicing can be part of adaptive stress responses in certain cell organelles, such as the endoplasmic reticulum (ER). Moreover, abnormal mRNA splicing could also be related to cell apoptosis. Under stress conditions, unfolded proteins accumulate in the ER and form aggregates. These abnormal agglomerations engage a response process called unfolded protein response (UPR), which is triggered thanks to a few different stress sensors that reside in the ER. One of those sensors is inositol-requiring enzyme 1α (IRE1α), a type I transmembrane protein, which once activated, initiates the abnormal splicing of the mRNA that encodes the transcription factor X-box binding protein 1 (XBP1), leading to the translation of a more stable spliced form of XBP1 (XBP1s). XBP1s translocates to the nucleus, where it controls the upregulation of a subset of UPR-related genes linked to protein folding, quality control, ERAD and ER/Golgi biogenesis. Furthermore, prolonged ER stress leads to the inactivation of IRE1α signaling, which in turn is associated with the attenuation of XBP1 mRNA splicing, process that could sensitize cells to apoptosis.
In the heat-shock protein 47 (HSP47), the selection of the 5’ splice-site in the non-coding region of the pre-mRNA is performed more efficiently. In cold shock, alternative pre-mRNA splicing is induced in neurofibromatosis type 1 (NF1) which brings about a cryptic exon. Stress induced long-term neuronal hypersensitivity is associated with stress-induced alternative splicing of the pre-mRNA of neuronal acetylcholinesterase (ACHE).

### Impact of Heat Shock Stress

Many types of stress, including heat shock, can immediately block many crucial metabolic processes such as DNA replication until recovery. Heat shock proteins (HSPs) help protect cells from injury and aid cell recovery and after heat shock conditions subside. The blocking of pre-mRNA splicing in heat-shocked proteins is well characterized. HSPs are not affected in their expression, however, because they do not contain any introns.
SRp38 is an SR protein, that when overexpressed, antagonizes the activity of SR protein SF2/ASF (splicing factor 2/alternative splicing factor). SRp38 is unique in that, when phosphorylated, it activates sequence-specific splicing that requires an as of yet unidentified cofactor. This activity stems from SRp38’s entry into a complex with U1 and pre-mRNA which strengthens the interaction of U1 and U2 with pre-mRNA. SRp38 is a strong splicing repressor when dephosphorylated after heat shock; after mild heat shock it is rephosphorylated, accompanying the return of splicing activity.
Nuclear stress bodies (nSBs) are proposed to control splicing activity under stress by bringing a set of splicing factors to the region where they bind to SATIII transcripts. The nSBs are the sites of accumulation of heat-shock factor 1 (HSF1) in human cells, and appear fleetingly after mild heat shock, chemical and hypertonic stresses. They are also the site of accumulation of pre-mRNA splicing factors (SF2/ASF, 9G8, SRp30c for the adenoviral E1A gene). The nSBs are assembled on regions of chromatin that consist of long satellite III (SatIII) DNA. After heat shock, chromatin reorganization occurs along with HSF1 transcription of SatIII RNAs. Recruitment of the SF2/ASF and SRp30c proteins requires the stress-induced SATIII transcripts. Reducing the transcription blocks the SR protein splicing factor recruitment.

### Structural Insights into RNA Splicing

The study of introns and alternative splicing has lead to the additional classification for introns in both eukaryotes and prokaryotes. Two groups of introns have been discovered. The first group of introns, Group I, were the first self-splicing ribozymes to be discovered. Group II introns were later reported. The Group II intros have highly complex RNA structures and they also possess a unique diverse range of chemical reactivity. The Group II intros possess the capability to catalyze the 2'-5' bond formation, and the ability to retrotranspose onto DNA. Retrotranspositions onto DNA requires the help of intron-encoded proteins. Specific analysis of Group II's secondary structure revealed six structural domains. Domain V is the mos conserved phylogenetically (closely related among various groups of organisms). The lower helix of Domain V possess a catalytic triad that consists of nucleotides that is very similar to that of a spliceosome called U6 spliceosomal RNA. This has led to the belief that Group II introns share acommon ancestor with nuclear introns and the eukaryotic splliceosome. This has led to further meticulous study of Group II introns.

### Study of Group II Introns

Group II introns have been further classified into three main structural elements based on their RNA secondary sturcture. The three groups are IIA, IIB, and IIC. Analysis by reverse transcriptases have shown that group IIC introns are the most primal and simplistic lineage of the Group II introns. The secondary structure of lineage IIC is much simple than that of IIA and IIB. Because the Group IIC are much smaller and simplistic, they are much more preferred by study of crystallization. Oceanobacillus iheyensis was the first organism to have its Group II intron successfully crystallized and examined via x-ray diffraction. Group IIC is also significantly different from Group IIA and IIB by the fact that the nucleophile used during the first step of the splicing is a water molecule. Group IIA and IIB use a 2'-OH nucleophile from the adenosine in Domain VI. Because Group IIC uses a water molecule the introns released are linear molecules, while Groups IIA and IIB introns will be released as a lariat branched species. The use of the Group IIC intron has further suggest that the active site is composed of the bulge and catalytic triad of Domain V, mentioned above. It has been shown that these regions are influenced by the binding of catalytic metal ions, i.e. magnesium. This meatal ion interaction is very common in protein in order to influence shape, i.e. Fe hemoglobin. The ion helps modulate the eclectrostatic environment at the core of the intron or protein. The metal ion interaction can also make or breack the phosphodiester linkages in the DNA and RNA polymerases.

### Possible Splicing Errors

Although the rate of splicing errors is very low, it does happen occasionally due to several distinct possibilities. A splicing error will most likely result in a mutation of a splice site and could compound into losing the function of the particular site. An exposure of a premature stop codon or a misplace/misinserted exon or intron could all lead to a mutation. A mutation from variations in the splice location which could cause a wrong amino acid to be interpreted. A misinterpreted amino acid could result in reducing specificity. All mutations could result in wrongly constructed proteins, which can be life threatening, i.e. cickle cell anemia or cystic fombrosis. Fortunately many splicing errors can be safeguarded by Nonsense-mediated mRNA decay.

### Nonsense-Mediated Decay

Nonsense mediated decay is the cellular mechanism of mRNA that exists to detect incorrectly spliced information, and to prevent the expression of incorrect proteins. After transcription the mRNA will reassemble with ribonucleoprotein. Nonsense mediated decay is initiated by exon junction comples that are cut out from the genetic information during the mRNA processing. Exon junction complexes located past a nonsense codon act as tags for the mRNA ribonucleoprotein, RNP. The RNP is able to recognize disorganizantion and wrong splicing from the pressence of these exons that were supposed to be cut out.The nonsense mediated decay will transport the excon tagged misinformation set out the nucleus and into the cytosol where the misinformation RNA is degraded.

## References

Cellular stress and RNA splicing. Biamonti G, Caceres JF. Trends Biochem Sci. 2009 Mar;34(3):146-53. Epub 2009 Feb 7.
William Fontaigne De La Tour Dautrieve
Aaron A. Hoskins, Melissa J. Moore, The spliceosome: A Flexible, Reversible Macromolecular Machine, Trends in Biochemical Sciences, Volume 37, Issue 5, May 2012. <http://www.sciencedirect.com/science/article/pii/S0968000412000345>
Sharp, Phillip A. "The Discovery of Split Genes and RNA Splicing." Trends in Biochemical Sciences 30.6 (2005): 279-81.
Woehlbier, U., Hetz, C. Modulating stress responses by the UPRosome: A matter of life and death. Trends in Biochemical Sciences, June 2011, Vol. 36, No. 6.