RNA Splicing

by Kevin Ahern, PhD

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    00:01 Now splicing is an important process and I should emphasize that splicing occurs almost exclusively in eukaryotes.

    00:08 There is a only a couple of genes known anywhere in the prokaryotic kingdom where splicing actually occurs.

    00:15 Splicing occurs on almost all types of RNA found in eukaryotic cells.

    00:21 But mostly commonly we find it in messenger RNA.

    00:24 It is found in some tRNAs and in some ribosomal RNAs but most commonly we find it in messenger RNAs.

    00:31 Now splicing, as I said, involves the removal of sequences internal to a pre-RNA, in this case a pre-mRNA.

    00:39 This removal of sequences takes out sequences in the RNA that we call introns.

    00:47 So thinking back to the original DNA, the original DNA had a sequence that was transcribed and that DNA sequence contain intron sequences.

    00:56 The introns in the RNA that's produced get removed and so that's what's being depicted on the screen.

    01:04 We see the removal of the introns by a structure called the spliceosome.

    01:08 And the spliceosome, as we will see, is a complex of RNA and proteins that performs this function.

    01:14 The removal of the intron results in production of a molecule we call a lariat structure.

    01:21 And all that really means is that, that intron had one end of itself bound to another part of itself to make a sort of loop structure, as you see on the image, that has a form of like that of a lariat.

    01:32 The spliceosome is a complex of both RNAs and protein. The RNAs are called snRNAs; because, they correspond to what we refer to as small nuclear RNAs.

    01:45 And when complex to proteins we called them small nuclear ribonucleoproteins or S-N-R-N-Ps.

    01:54 Some people call those snrnps although that's probably not exactly an accurate pronunciation.

    01:58 Now this image depicts the sort of chemical way in which this process occurs.

    02:04 We can see for example the pre-messenger RNA at the top.

    02:09 It has an exon on the left and an exon on the right. Now I need to tell you what those terms are.

    02:13 The exons are the parts that get joined in the splicing process.

    02:19 Intron is the part that gets removed.

    02:21 Now it's very important that this process proceed precisely; because, if the splicing is made one nucleotide or another in the wrong direction then that will also have significant effects because it will affect the genetic code that is coming in the mRNA that's being translated.

    02:41 So this process must occur accurately if the protein that is going to be made from this mRNA is itself going to be made properly.

    02:50 So there are couples of things that we see in introns sequences and you can see them labelled above that are common among almost all introns.

    03:00 At the 5 prime end of the intron sequence, we see a sequence of GU that almost always occurs and probably helps to make sure that the orientation and the removal of the intron happens at the same place every time.

    03:16 At the 3 prime end of the intron, we see a sequence of AG and that almost always also occurs within introns.

    03:24 Then about 30 nucleotides or so ahead of that AG, there is a sequence that is an A, as you can see here, and that A is usually adjacent to a sequence of pyrimidines that are not shown on this figure.

    03:40 Now in this splicing process what happens is the A that you see on the image makes a nucleophilic attack on the G at the 5 prime end of the intron.

    03:51 That nucleophilic attack results in the covalent bonding between the G and the A, as you can see here and we have started to form the intron.

    04:02 The structure gets resolved when the joining of the exon on the left occurs with the exon on the right.

    04:11 That results in this cutting of the bond between the G on the right and the exon.

    04:19 That results in the removal of the lariat structure that you can see and the production of the spliced messenger RNA here.

    04:28 Now common intron sequences help to orient and make this process possible and without these common intron sequences the cell would have no way of knowing where to make the cut.

    04:37 Now one other thing I didn't note about the lariat structure is that it has an unusual bond in it.

    04:45 You recall from the structure of nucleic acid that the nucleotides are joined together in a 5 prime to 3 prime orientation such as the 3 prime end of one nucleotide is covalently bonded to the 5 prime structure of the next one.

    05:03 So that 5 prime 3 prime, 5 prime 3 prime carries all the way through an RNA and a DNA.

    05:08 But this bond right here is unusual in that. Instead of having a 5 prime-3 prime bond, it has a 5 prime-2 prime bond.

    05:17 And that's because the 2 prime hydroxyl is available and it's what making the nucleophilic attack that I mentioned.

    05:23 It's an unusual structure. But this is the one place in biology where it occurs.

    05:28 The excised inton comes out as a lariat, as I said, and that lariat can also be used for other things.

    05:37 In a few cases the lariat itself actually contains another gene, that's rather unusual.

    05:41 Or the lariat can simply be degraded and the base is used inside of that structure.

    05:47 Now this figure shows the joining of the snRNPs to the inton for making this process happen.

    05:54 You can see in this process that U1 is added first, U2 is added second and then the complex of U4, U5, and U6 are added third.

    06:05 Now we are not gonna look up close in personal at that individual structure but if I set to say that's there is very precise and specific structure that's created that is known as the spliceosome.

    06:15 That spliceosome some people has compared to ribosome in the sense that they both contain proteins and they both contain RNAs and they both perform functions on an RNA.

    06:25 Well, this one isn't performing translation. But this one is now excising introns.

    06:31 The snRNPs and the snRNAs that they contain appear to play a very important role in aligning the A that's going to make the nucleophilic attack on the G in the right orientation.

    06:46 So once that orientation is setup then that excision of the intron and the formation of a lariat structure can actually occur.

    06:53 So there is actually a sequential series of events that happen in this process.

    06:59 After the structure has formed we see removal of couple the snRNPs, the U4, the U1 for example and then released of the others and finally the spliced messenger RNA along with a lariat structure.

    About the Lecture

    The lecture RNA Splicing by Kevin Ahern, PhD is from the course RNA and the Genetic Code.

    Included Quiz Questions

    1. It is almost exclusively found in prokaryotic cells
    2. It involves the removal of intervening sequences called introns from an RNA
    3. It involves the joining together of sequences called exons that are separated by introns
    4. It creates lariat structures in the excised introns
    1. An unusual 5’ to 2’ bond
    2. An unusual 2’ to 3’ bond
    3. An unusual 3’ to 5’ bond
    4. An unusual 3’ to 1’ bond
    5. An unusual 1’ to 5’ bond
    1. During self-splicing of pre-mRNA, the exons get joined to each other in a lariat structure form, whereas the intron gets released as a linear molecule
    2. A spliceosome cuts the introns, having a GU sequence at the 5’ end (splice donor site) and AG sequence at the 3’ end (splice acceptor site) in the pre-mRNA
    3. The branch site located 20-50 bases upstream of the AG sequence contains a consensus sequence CU(A/G)A(C/U)
    4. Spliceosomal splicing involves two transesterification reactions: (1) Nucleophilic attack of 2’OH of A of branch site at the 5’ splice site; and (2) Nucleophilic attack of 3’OH of released 5’ exon at the first nucleotide of the intron at the 3’ splice site
    5. The spliceosome complex plays a crucial role in RNA splicing and is composed of U1, U2, U4, U5, and U6 snRNPs

    Author of lecture RNA Splicing

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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