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    RNA Splicing
    Experiments, Studies, Demonstrations & Background Information
    For Science Labs, Lesson Plans, Class Activities & Science Fair Projects
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    Experiments and Demonstrations

    • mRNA Splicing Demonstration - Essential Cell Biology [View Experiment]
    • mRNA Splicing Demonstration - Virtual Cell Animation Collection (VCAC) [View Experiment]
    • The generally expressed hnRNP F is involved in a neural-specific pre-mR splicing event [View Experiment]
    • Translation of globin RNA spliced in vitro [View Experiment]
    • Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1[View Experiment]
    • Analysis and design of RNA sequencing experiments for identifying RNA editing and other single-nucleotide variants [View Experiment]
    • Thesis: Data Structures and Algorithms for Analysis of Alternative Splicing with RNA-Seq Data [View Experiment]
    • Dissertation: Characterisation of heterogeneous nuclear ribonucleoprotein G (hnRNP G) and its role in splice site selection [View Experiment]
    RNA Splicing Background Information

    Definitions

    RNA splicing is the modification of messenger RNA (mRNA) after transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic mRNA before it can be used to produce a correct protein through translation.

    Exons are DNA regions within a gene that are translated into a protein and separated from each other by introns.

    An intron is a DNA region within a gene that is not translated into protein and resides between exons.

    Basics

    For many eukaryotic introns, splicing is done in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs), but there are also self-splicing introns.

    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.

    Spliceosomal introns often reside in eukaryotic protein-coding genes.

    Spliceosome formation and activity: Splicing is catalyzed by the spliceosome which is a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs, pronounced 'snurps' ). The RNA components of snRNPs interact with the intron and may be involved in catalysis. Two types of spliceosomes have been identified (the major and minor) which contain different snRNPs.

    Trans-splicing is a form of splicing that joins two exons that are not within the same RNA transcript

    Self-splicing occurs for rare introns that form a ribozyme, performing the functions of the spliceosome by RNA alone. There are three kinds of self-splicing introns, Group I, Group II and Group III. Group I and II introns perform splicing similar to the spliceosome without requiring any protein. This similarity suggests that Group I and II introns may be evolutionarily related to the spliceosome. Self-splicing may also be very ancient, and may have existed in an RNA world present before protein. Although the two splicing mechanisms described below do not require any proteins to occur, 5 additional RNA molecules and over 50 proteins are used and hydrolyzes many ATP molecules. The splicing mechanisms use ATP in order to accurately splice mRNA's. If the cell were to not use any ATP's, the process would be highly inaccurate and many mistakes would occur.

    tRNA splicing is another rare form of splicing that usually occurs in tRNA. The splicing reaction involves a different biochemistry than the spliceomsomal and self-splicing pathways. Ribonucleases cleave the RNA and ligases join the exons together.

    Evolution: Splicing occurs in all the kingdoms or domains of life, however, the extent and types of splicing can be very different between the major divisions. Eukaryotes splice many protein-coding messenger RNAs and some non-coding RNAs. Prokaryotes, on the other hand, splice rarely and mostly non-coding RNAs. Another important difference between these two groups of organisms is that prokaryotes completely lack the correct spliceosomal pathway. Because spliceosomal introns are not conserved in all species, there is debate concerning when spliceosomal splicing evolved. Two models have been proposed: the intron late and intron early models.

    Biochemical mechanism: Spliceosomal splicing and self-splicing involves a two-step biochemical process. Both steps involve transesterification reactions that occur between RNA nucleotides. tRNA splicing, however, is an exception and does not occur by transesterification.

    Alternative splicing is a process by which the exons of the RNA produced by transcription of a gene (a primary gene transcript or pre-mRNA) are reconnected in multiple ways during RNA splicing. The resulting different mRNAs may be translated into different protein isoforms; thus, a single gene may code for multiple proteins.

    Experimental manipulation of splicing: Splicing events can be experimentally altered by binding steric-blocking antisense oligos such as Morpholinos or Peptide nucleic acids to snRNP binding sites, to the branchpoint nucleotide that closes the lariat, or to splice-regulatory element binding sites.

    Splicing Common errors:

    • Mutation of a splice site resulting in loss of function of that site. Results in exposure of a premature stop codon, loss of an exon, or inclusion of an intron.
    • Mutation of a splice site reducing specificity. May result in variation in the splice location, causing insertion or deletion of amino acids, or most likely, a loss of the reading frame.
    • Displacement of a splice site, leading to inclusion or exclusion of more RNA than expected, resulting in longer or shorter exons.

    Protein splicing: Not only pre-mRNA but also proteins can undergo splicing. Although the biomolecular mechanisms are different, the principle is the same, that parts of the protein, called inteins instead of introns, are removed. The remaining parts, called exteins instead of exons, are fused together. Protein splicing has been observed in all sorts of organisms, including bacteria, archaea, plants, yeast and human.

    Topics of Interest

    An exon is a nucleic acid sequence that is represented in the mature form of an RNA molecule after either portions of a precursor RNA (introns) have been removed by cis-splicing or by two or more precursor RNA molecules have been ligated by trans-splicing. The mature RNA molecule can be a messenger RNA or a functional form of a non-coding RNA such as rRNA or tRNA. Depending on the context, exon can refer to the sequence in the DNA or its RNA transcript.

    An intron is a DNA region within a gene that is not translated into protein. These non-coding sections are transcribed to precursor mRNA (pre-mRNA) and some other RNAs (such as long noncoding RNAs), and subsequently removed by a process called splicing during the processing to mature RNA. After intron splicing (ie. removal), the mRNA consists only of exon derived sequences, which are translated into a protein.

    A spliceosome is a complex of specialized RNA and protein subunits that removes introns from a transcribed pre-mRNA (hnRNA) segment. This process is generally referred to as splicing.

    The minor spliceosome is a ribonucleoprotein complex that catalyses the removal (splicing) of an atypical class of spliceosomal introns (U12-type) from eukaryotic messenger RNAs in plant, insects, vertebrates and some fungi (Rhizopus oryzae). This process is called noncanonical splicing, as opposed to U2-dependent canonical splicing. U12-type introns represent less than 1% of all introns in human cells. However they are found in genes performing essential cellular functions.

    The exon junction complex (EJC) has major influences on translation, surveillance and localization of the spliced mRNA. It is first deposited onto mRNA during splicing and is then transported into the cytoplasm. There it plays a major role in post-transcriptional regulation of mRNA. It is believed that exon junction complexes provide a position-specific memory of the splicing event. The EJC consists of a stable heterotetramer core, which serves as a binding platform for other factors necessary for the mRNA pathway. The core of the EJC contains the protein eukaryotic initiation factor 4AIII (eIF4AIII; a DEAD-box RNA helicase) bound to an adenosine triphosphate (ATP) analog, as well as the additional proteins Magoh and Y14. In order for the binding of the complex to the mRNA to occur, the eIF4AIII factor is inhibited, stopping the hydrolysis of ATP. This recognizes EJC as an ATP dependent complex.

    Precursor mRNA (pre-mRNA) is an immature single strand of messenger ribonucleic acid (mRNA). pre-mRNA is synthesized from a DNA template in the cell nucleus by transcription. pre-mRNA comprises the bulk of heterogeneous nuclear RNA (hnRNA). The term hnRNA is often used as a synonym for pre-mRNA, although strictly speaking hnRNA may include nuclear RNA transcripts that do not end up as cytoplasmic mRNA.

    Source: Wikipedia (All text is available under the terms of the GNU Free Documentation License and Creative Commons Attribution-ShareAlike License.)

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