Biochemistry 3107 - Fall 2002

Introns

The Discovery of Introns

In 1977, research groups working on the expression of adenovirus late genes at MIT and at Cold Spring Harbor reported that the mRNAs were:

"mosaic molecules consisting of sequences complementary to several non-contiguous segments of the viral genome".

Quote from Adenovirus amazes at Cold Spring Harbor (1977) Nature 268: 101-104.

 

Phil Sharp (at MIT) and Rich Roberts (at Cold Spring Harbor) led the research groups which made this discovery. They shared the Nobel Prize in Medicine (1993) for their discovery.

The structures of the "mosaic" received its name from Walter Gilbert in 1978:

The notion of the cistron, the genetic unit of function that one thought corresponded to a polypeptide chain, now must be replaced by that of a transcription unit containing regions which will be lost from the mature messenger -- which I suggest we call introns (for intragenic regions) -- alternating with regions which will be expressed -- exons. The gene is a mosaic: expressed sequences held in a matrix of silent DNA, an intronic matrix.

Gilbert, W. (1978) Why genes in pieces? Nature 271: 501

 

By this time, it had become clear that the occurence of introns was not restricted to viral mRNAs but was a general feature of eukaryotic mRNAs. A number of research groups working in the field of eukaryotic transcription had, in fact, been close on the heels of Sharp and Roberts with respect to the discovery of introns. Early in 1978, Science reported that spacer sequences (i.e. introns) had been identified in genes for b-globin, immunoglobulin, ovalbumin, tRNA and rRNA.

Pierre Chambon's group in Strasbourg demonstrated the existence of introns in the ovalbumin gene. This intron/exon structure of this gene is used as the typical example in many texts.

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Chambon focused on the ovalbumin gene because it was easy to obtain a plentiful supply of ovalbumin mRNA. Chick oviduct cells synthesize almost nothing but ovalbumin and they contain one principal mRNA as a result. Chambon hybridized ovalbumin mRNA with the DNA fragment that coded for the gene and examined the results in the electron microscope (this is an example of the technique called R-looping). The results were quite clear. 7 single-stranded regions were seen that looped out of the double-stranded RNA:DNA hybrid. These correspond to the 7 introns in the gene. A small single-stranded regions was also observed at the 3' end - we now know that this corresponds to the poly(A) tail on the mRNA.

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HnRNA processing

The initial nuclear RNA copy of protein-coding genes transcribed in eukaryotes is known as hnRNA - heterogeneous nuclear RNA so called because of its large size. hnRNA is processed while it is being synthesized and before it leaves the nucleus en route to the ribosomes located in the cytoplasm. Three types of modification are made:

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• The poly(A) tail is added to the 3' end.
  
  
• A cap is added to the 5' end.
  
  
• Introns are spliced out.

 

HnRNA had been detected at least 10 years previously but its significance had not been realised. Since hnRNA was considerably larger than any mRNA needed to be in order to code for any known protein, hnRNA had not been considered to have a direct role in gene expression. The discovery of introns solved the mystery of hnRNA.

Many genomes, especially plant genomes, are very much larger than expected. The discovery of introns also helped to explain the size of eukaryotic genomes. An extreme example is that of the dystrophin gene. The initial pre-mRNA transcript could be > 2 million nt in size but this is spliced down to an mRNA of 14,000 nt by the removal of 78 introns!

In addition to introns, eukaryotic genomes are full of repetitive DNA elements. Recently, it has been found that 50% of the maize genome consists of nested retroviral elements. All told, 60 - 80% of the maize genome is made up of repetitive sequence; the retroviral repeats make up a huge fraction of the genome.

Classes of Introns

Four major classes of introns have been distinguished based on their mechanisms of splicing:

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Group I Self-Splicing Introns

These introns are found primarily in nucleolar rRNA genes and in organelle genomes -- both in mitochondrial DNA (mtDNA) and in chloroplast DNA (ctDNA). No additional proteins and no energy source is required for the splicing reaction. However, a free guanosine nucleoside is required as the catalytic agent in the mechanism.

 

Group II Self-Splicing Introns

This type of intron is found primarily in mtDNA and in ctDNA. As with Group I introns, no additional proteins and no energy source is required for the reaction. The catalytic agent is an internal hydroxyl group within the intron.

 

Nuclear mRNA Spliceosomal Introns

The mechanism of the splicing reaction in nuclear mRNA introns is similar to that of the Group II Introns, splicing of these introns requires the participation of a specific set of protein-RNA particles.

 

Nuclear tRNA Enzymatically Spliced Introns

These introns also require the help of enzymes to catalyze their removal but the mechanism is completely different - being a cut and rejoin type of mechanism.

Introns & Evolution

The discovery of introns led to a search to see how prevalent they are. Introns are widespread in eukaryotes but they are quite rare in prokaryotes. This has prompted much speculation with regards to the evolution of organisms in general and the role introns may have played in that evolution.

There are two schools of thought:

Introns-early

Proponents of this school argue that introns were an essential feature of the earliest organisms. Their absence in bacteria, they argue, is due to the fact that that the shorter division times of bacterial cells means that bacteria have had many more growth cycles in which to evolve. This evolution has brought about the loss of nearly all ancestral introns.

 

Introns-late

Proponents in this school argue that the earliest organisms did not contain introns. They argue that introns are a relatively recent arrival in the eukaryotic lineage that has been necessary to help generate the diversity of regulatory mechanisms that are required to control gene expression in multicellular highly differentiated organisms. In this view, prokaryotes do not have introns because they never had them in the first place.

 

The controversy and the arguments shift back and forth as new research contributes to the debate.