Biochemistry 3107 - Fall 2002

Splicing

 

The Discovery of Self-Splicing RNA

Tom Cech (right) discovered self-splicing in rRNA in 1981/82. The discovery was important for two reasons.

 

  • First, it shattered the long-held belief that only proteins could catalyze biological reactions.

  • Second it provided the mechanistic details for RNA splicing in at least one system.

Cech was awarded the Nobel Prize in Chemistry in 1989 along with Sidney Altman (right), who discovered that the active catalytic component of Ribonuclease P in E. coli was an RNA molecule.

 

The following description of Cech's discovery of self-splicing is based on his Nobel Prize address (which is referenced in the Resource material, below).

 

In 1978, when he set up his own lab in Boulder, Colorado, Cech had decided that he wanted to study the structure and expression of a single gene. He chose the rDNA gene (i.e. the gene that codes for rRNA) of Tetrahymena. In ciliated protozoa, such as Tetrahymena, transcription takes place in the macronucleus, which contains gene-sized pieces of DNA. In the case of rDNA, the macronucleus contains 10,000 copies of the rDNA genes.

 
 cartoon and photo from Brian Harmon's homepage at UCSF

 

The attraction for Cech was not the abundant supply of rDNA as much as the ready supply of all the factors that were necessary for its transcription. He set out to identify and purify these.

His first step was straightforward and a typical step for any biochemist seeking to dissect the components of a cellular process. He tried to see if he could observe transcription in vitro using a crude cell-free system consisting of:

 

  • Isolated nuclei from Tetrahymena thermophila.
    These provide both the RNA polymerase and the rDNA.

  • All four nucleoside triphosphates, one of which was radioactively labelled.

  • A standard transcription buffer with suitable concentrations of salt.

  • a-amanitin.
    This suppressed all transcription except that by RNA polymerase I - in other words all transcription except that of rDNA genes.

His results on the one hand were just as he expected them to be and on the other hand, intriguing. He observed synthesis of a high molecular weight species of RNA. This had the expected size (26S) for pre-rRNA. However, he also observed a smaller 9S RNA. This RNA accumulated with reaction time. He thought at first that it must be a by-product of the processing reactions that were required to generate mature rRNA from the precursor pre-rRNA, i.e. that it might be the 5' external transcribed spacer sequence or the RNA corresponding to the region between the 17S and 5.8S rRNAs.

[MVH28-20] [rDNA.GIF]

He asked his student, Art Zaug, to identify the unexpected RNA. To their surprise, it was found to be the intervening sequence (intron) that was known to occur within the 26S gene.

This was an exciting finding! The fact that Cech's crude system seemed to splice out the intron in vitro meant that he had discovered a system with which he could dissect the biochemical mechanism of splicing. Only one other such system - for a tRNA - was known at that time. Cech reckoned that, since each cell had 10,000 copies of the rDNA gene each of which was expressed, and therefore spliced, at the rate of 1 rRNA copy per second, then his nuclei should be an abundant source of the splicing enzyme.

To set about characterizing the system, they first of all had to find conditions in which pre-rRNA synthesis occurred but splicing did not. This was straightforward and it allowed them to isolate the pre-rRNA as a substrate. Then they set up a test reaction.

Their reaction was simple. In one tube they incubated the substrate pre-rRNA and nuclear extract in the same reaction buffer that was used for their transcription reactions and in which they had seen splicing. In a second tube - which was their control - they left out the nuclear extract.

The results both pleased and surprised them! The reaction worked; they observed splicing in vitro. However, they also saw splicing in their control tube, the one in which no nuclear extract was present. In the words of Cech:

 

"Well Art, this looks very encouraging, except you must have made some mistake making up the control sample."

 

How often supervisors utter words such as these to their students! How rare it is that they are so wrong! Zaug had made up his buffers correctly.

 

Their next step was to sequence the intron RNA to verify that it was indeed the correct RNA and that it was being spliced correctly. When this was done, they found that their sequence matched the known sequence exactly except for the presence of a Guanine nucleotide at the 5' end. Thinking that the previously determined sequence must be wrong they called the Joe Gall laboratory, who determined the sequence, to point this out:

 

We telephoned them (the Joe Gall laboratory), advising them that they had determined most of the sequence correctly but had apparently missed one G right at the 5' end of the IVS (intron). Much to our surprise, they defended every nucleotide of their sequence: no ambiguity in the DNA sequence, at least in that region, and no chance of a G at the 5' splice site.

 

In the meantime, Cech was trying to refine the components that were required for the in vitro splicing reaction. He had found that removal of three of the four NTPs had no effect on the reaction. However, the fourth, GTP was absolutely required. Cech wondered whether it was a coincidence that the extra nucleotide at the 5' end of the spliced intron was the same as that required for the reaction to proceed in vitro?

 

The obvious hypothesis was that GTP was required so that it could be added to the 5' end of the IVS (intron) during splicing. The test was simple: mix 32P-labelled GTP with unlabelled pre-rRNA, and look for labelling of the IVS (intron) RNA concomitant with its excision. The experiment was the strangest I had ever performed. On the one hand, its success was a straightforward prediction from our existing knowledge of the system. On the other hand, it seemed incredibly naive and unrealistic to expect that simple mixing of a nucleotide with phenol-extracted proteinase-treated RNA could possibly result in the formation of a covalent bond. I certainly didn't want to be embarrassed in front of my graduate students and colleagues by the failure of such an experiment, so I did it very quietly.

 

The rest is, as they say, history. The experiment worked. And, after further confirmation and characterization, they were certain. Splicing of the intron required no protein - just the intron itself and guanosine.

 

Cech coined the term ribozyme to describe RNA molecules with a catalytic activity. He received some grief about this since his self-splicing intron did not possess true catalytic activity; it catalyzed its own excision but not that of other molecules. The demonstration by Sidney Altman that the M1 RNA component of RNase P is the catalytic agent, as well as a number of other more recent examples (e.g. peptidyl transferase) have overcome any objections to the term.

 

Mechanisms of Intron splicing

Recall that there are four major classes of introns:

[S33-47]

 

Introns in the first three classes are spliced via two transesterification reactions. In all cases, one phosphodiester bond is exchanged for another so there is no energetic cost to the reaction. The different classes can be distinguished on the basis of the initiating nucleophilic attack agent and whether additional proteins are required or not.

 

Group I Intron Splicing

The basic steps of the mechanism are as follows:

[25-23] [S33-40]

 

 

The reaction products are a correctly spliced RNA and a linear intron with a G at the 5' end.

It is important to keep in mind that the self-splicing of introns, such as the Tetrahymena rRNA intron, depends on the overall tertiary structure of the intron. Even though we often think of them as single stranded, RNA molecules have very definite secondary and tertiary structure. Sequence variability can be permitted as long as certain structural features are maintained. In the case of the pre-rRNA intron, it folds up such that the 5' and 3' splice sites are quite close together with the catalytic guanosine nucleotide bound nearby.

[25-24] [image]

 

Group II Intron Splicing

 

 

 

Diagrams from Melissa Moore's Home Page

The basic steps of the mechanism are as follows:

[Lod12-20]

 

Since one phosphodiester bond has been replaced with another, this is still a transesterification reaction.

The 3' end of the upstream exon is now free.

 

This exchanges the intron-exon phosphodiester bond for an exon-exon phosphodiester bond and is the second transesterification step.

 

The reaction products are a correctly spliced RNA and an intron with an intramolecular 2'->5' phosphodiester which forms a lariat structure. As with group I self-splicing, a correct secondary and tertiary structure is required.

[Lod12-27]

Note that the differences between Group I and Group II self-splicing reactions are:

 

 

Spliceosomal Intron Splicing

The basic steps of the mechanism are the same as those for Group II Self-Splicing Introns:

[25-20] [MVH28-32] [Image]
(Image above and Table below from D. A. Brow (2002) Allosteric Cascade of Spliceosome Activation. Annu. Rev. Genet. 2002. 36:333­60)

 

Again, the adenine nucleotide is located within the intron and the reaction generates a nucleotide which simultaneously participates in three phosphodiester bonds. Since one phosphodiester bond has been replaced with another, this is still a transesterification reaction.

The 3' end of the upstream exon is now free.

This exchanges the intron-exon phosphodiester bond for an exon-exon phosphodiester bond and is the second transesterification step.

The reaction products are a correctly spliced RNA and an intron with a lariat structure.

 

Unlike the Group II Self-splicing introns, this reaction absolutely requires the participation of an elaborate assembly of other factors. These are the small nuclear ribonucleoprotein particles or snRNPs.

Each snRNP is a complex of many proteins [Table] and a small stable U-RNA called a small nuclear RNA or snRNA. Five of them are involved in nuclear mRNA splicing:

[Lod12-21]

 

snRNA complementarity function
U1 5' end of the intron recognizes and binds the 5' splice region
U2 Branch point recognizes and binds the branch point region. Also pairs with the U6 snRNA during spliceosome assembly
U4 U6 snRNA binds to and inactivates U6. During assembly of the spliceosome, the U4-U6 interaction is unpaired and replaced with an interaction between U2 and U6. U6 also replaces the U1 interaction with the 5' splice junction.
U5 upstream and downstream exons binds to both exons keeping them in the spliceosomal complex.
U6 U4 (and U2) binds with U2 in the assembled spliceosome.

 

The assembly of a spliceosome depends on the recognition of specific sequence elements in the intron by individual snRNPs. In general nuclear mRNA introns contain the conserved elements:

GU----------A----AG

[25-19] [Lod12-18]

Tom Schneider's logo representation provides an alternative perspective:

 

The assembly of the spliceosome proceeds as follows:

[MVH28-33] [Lod12-24]

  • The 5' splice junction is recognized by the U1 snRNP.

  • The 3' splice junction is recognized by the U2AF (U2 snRNP associated) factor. The U1 snRNP may also recognize sequences at the 3' splice junction.

  • At the same time the Branch point is recognized by the U2 snRNP. Pairing between the U2 snRNA and the branch point sequence is not exact. A single adenine nucleotide is unpaired and forms a bulge thus exposing its critical 2' OH group for catalysis.

    [Lod23-3a]

  • The U4/U6 and U5 snRNPs enter the complex.

    [Lod23-3b]

  • U5 contacts the 5' exon sequences and, probably, it also contacts the 3' exon sequences later in the reaction. U1 is not be required after U5 has bound to the complex. Its interaction with the 5' splice region is replaced by U6.

  • The U4/U6 hydrogen bonds are unpaired and replaced with U2/U6 hydrogen bonds. U4 is no longer necessary for activity within the spliceosome and dissociates. U6 pairs with the 5' splice region replacing U1.

    [S33-39] [Lod23-3c]

  • Since U6 is the most highly conserved of all of the U snRNAs, it is believed to be central to the catalytic mechanism.

However, there is still some uncertainty about the number and nature of steps in splicosome assemble - see this figure.

This figure - which I forgot to show in lecture - illustrates the connection between transcription and splicing and offers a nice view of snRNA interactions with intermediate stages of the splicing reaction.

The critical role of the U6 snRNA in the assembly of a catalytically active spliceosome is shown in the following picture:

[Lod12-23c]

 

Note how the U6 snRNA is maintained in an inactive form by the U4 snRNA. The spliceosome is activated once U6 has paired both with the U6 snRNA and with the 5' splice junction region.

 

 

Splicing reactions are the principal reactions which involve ribonucleoprotein particles in the cell. However, other reactions are known in which other ribonucleoprotein particles participate.

Some of the other snRNPs that are known are:

 

 

Nuclear tRNA Enzymatically Spliced Introns

tRNAs also contain introns. Some, such as those in cyanobacteria are Group I self-splicing introns. Others, such as those in yeast, rely on a separate enzyme-based system for removal of the introns.

 


RESOURCE MATERIAL
VOET, VOET & PRATT
  1. Chapter 25, Transcription and RNA Processing, pages 822 - 830
  2. Chapter 27, Regulation of Gene Expression, pages 912 - 914
STRYER
  1. Chapter 33, RNA Synthesis and Splicing, pages 860-867
  2. Chapter 33, RNA Synthesis and Splicing, pages 869-870
LEHNINGER
  1. Chapter 25, RNA Metabolism, pages 866 - 870
  2. Chapter 25, RNA Metabolism, pages 877 - 879
TAMARIN
  1. Chapter 10, pages 255 - 263
WEB SITES
RESEARCH PAPERS
  1. Self-splicing and Enzymatic Activity of an Intervening Sequence RNA from Tetrahymena (Nobel Lecture) by Thomas R. Cech is the author's own account of the work that led to his award of the Nobel Prize in Chemistry in 1989. It is published in Angew. Chem. Int. Ed. Engl. 29: 759-768 (1990).

Format and Original Material © Martin E. Mulligan, 1996-2002