RNA Splicing: Introns, Exons and Spliceosome

By:Suzanne Clancy, Ph.D.2008Nature Education

Citation:Clancy,S.(2008)RNA splicing: introns, exons and spliceosome.Nature
Education1(1):31

What's the difference between mRNA and pre-mRNA? It's all about splicing of introns. See
how one RNA sequence can exist in nearly 40,000 different forms.

For most eukaryotic genes (and some prokaryotic ones), the initial RNA that is transcribed from a gene's
DNA template must be processed before it becomes a mature messenger RNA (mRNA) that can direct the
synthesis of protein. One of the steps in this processing, called RNA splicing, involves the removal or
"splicing out" of certain sequences referred to as intervening sequences, or introns. The final mRNA thus
consists of the remaining sequences, called exons, which are connected to one another through the
splicing process. RNA splicing was initially discovered in the 1970s, overturning years of thought in the
field of gene expression.

Early Studies in Bacteria

Gene regulation was first studied most thoroughly in relatively simple bacterial systems. Most bacterial
RNA transcripts do not undergo splicing; these transcripts are said to be colinear, with DNA directly
encoding them. In other words, there is a one-to-one correspondence of bases between the gene and the
mRNA transcribed from the gene (excepting 5 and 3 noncoding regions). However, in 1977, several groups
of researchers who were working with adenoviruses that infect and replicate in mammalian cells obtained
some surprising results. These scientists identified a series of RNA molecules that they termed "mosaics,"
each of which contained sequences from noncontiguous sites in the viral genome (Berget et al., 1977;
Chow et al., 1977). These mosaics were found late in viral infection. Studies of early infection revealed long
primary RNA transcripts that contained all of the sequences from the late RNAs, as well as what came to
be called the intervening sequences (introns).
Subsequent to the adenoviral discovery, introns were found in many other viral and eukaryotic genes,
including those for hemoglobin and immunoglobulin (Darnell, 1978). Splicing of RNA transcripts was then
observed in several in vitro systems derived from eukaryotic cells, including removal of introns from
transfer RNA in yeast cell-free extracts (Knapp et al., 1978). These observations solidified the hypothesis
that splicing of large initial transcripts did, in fact, yield the mature mRNA. Other hypotheses proposed that
the DNA template in some way looped or assumed a secondary structure that allowed transcription from
noncontiguous regions (Darnell, 1978).

How Splicing Occurs

The biochemical mechanism by which splicing
occurs has been studied in a number of systems and
is now fairly well characterized. Introns are removed
from primary transcripts by cleavage at conserved
sequences called splice sites. These sites are found
at the 5 and 3 ends of introns. Most commonly, the
RNA sequence that is removed begins with the
dinucleotide GU at its 5 end, and ends with AG at its
3 end. These consensus sequences are known to be
critical, because changing one of the conserved
nucleotides results in inhibition of splicing. Another
important sequence occurs at what is called the
branch point, located anywhere from 18 to 40
nucleotides upstream from the 3 end of an intron.
The branch point always contains an adenine, but it

Figure 1:Pre-mRNA splicing.

Splicing of a pre-mRNA molecule occurs in

is otherwise loosely conserved. A typical sequence
is YNYYRAY, where Y indicates a pyrimidine, N
several steps that are catalyzed by small
denotes any nucleotide, R denotes any purine, and A
nuclear ribonucleoproteins (snRNPs). After the
denotes adenine. Rarely, alternate splice site
U1 snRNP binds to the 5 splice site, the 5 end
sequences are found that begin with the dinucleotide
of the intron base pairs with the downstream
AU and end with AC; these are spliced through a
branch sequence, forming a lariat. The 3 end of
similar mechanism.
the exon is cut and joined to the branch site by
Splicing occurs in several steps and is catalyzed by
a hydroxyl (OH) group at the 3 end of the exon
small nuclear ribonucleoproteins (snRNPs,
that attacks the phosphodiester bond at the 3
commonly pronounced "snurps"). First, the presplice site. As a result, the exons (L1 and L2)
mRNA is cleaved at the 5 end of the intron following
are covalently bound, and the lariat containing
the attachment of a snRNP called U1 to its
the intron is released.
complementary sequence within the intron. The cut
1985 Nature Publishing Group Konarska, M. M. et al.
end then attaches to the conserved branch point
Characterization of the branch site in lariat RNAs produced by
region downstream through pairing of guanine and
splicing of mRNA precursors. Nature 313, 556 (1985). All rights
adenine nucleotides from the 5 end and the branch
reserved.
point, respectively, to form a looped structure known
Figure Detail
as a lariat (Figure 1). The bonding of the guanine and
adenine bases takes place via a chemical reaction
known as transesterification, in which a hydroxyl
(OH) group on a carbon atom of the adenine "attacks" the bond of the guanine nucleotide at the splice site.
The guanine residue is thus cleaved from the RNA strand and forms a new bond with the adenine.
Next, the snRNPs U2 and U4/U6 appear to contribute to positioning of the 5 end and the branch point in
proximity. With the participation of U5, the 3 end of the intron is brought into proximity, cut, and joined to
the 5 end. This step occurs by transesterification; in this case, an OH group at the 3 end of the exon
attacks the phosphodiester bond at the 3 splice site. The adjoining exons are covalently bound, and the
resulting lariat is released with U2, U5, and U6 bound to it.
In addition to consensus sequences at their splice sites, eukaryotic genes with long introns also contain
exonic splicing enhancers (ESEs). These sequences, which help position the splicing apparatus, are found
in the exons of genes and bind proteins that help recruit splicing machinery to the correct site. Most
splicing occurs between exons on a single RNA transcript, but occasionally trans-splicing occurs, in which
exons on different pre-mRNAs are ligated together.
The splicing process occurs in cellular machines called spliceosomes, in which the snRNPs are found
along with additional proteins. The primary variety of spliceosome is one of the most plentiful structures in
the cell, and recently, a secondary type of spliceosome has been identified that processes a minor category
of introns. These introns are referred to as U12-type introns because they depend upon the action of a
snRNP called U12 (the common introns described above are called U2-type introns). The role of U12-type
introns is not yet defined, but their persistence throughout evolution and conservation between
homologous genes of widely divergent species suggests an important functional basis (Patel & Steitz,
2003).

Self-Splicing and Alternative Splicing

Some RNA molecules have the capacity to splice
themselves; the initial discovery of this self-splicing
ability in the protozoan Tetrahymena thermophila was
recognized with the Nobel Prize in 1989. The selfsplicing introns found in T. thermophila are now
referred to as Group I introns; this class also
includes other protozoan ribosomal RNA genes,
some fungal mitochondrial genes, and some phage
genes. Group I introns all fold into a complex
secondary structure with nine loops and employ

Figure 2:A schematic representation of

transesterification reactions as described above. On

the other hand, Group II self-splicing introns are
found in mitochondrial genes and are excised by a
mechanism that bears similarities to pre-mRNA
splicing, including the production of lariats. For this
reason, it has been proposed that perhaps pre-mRNA
introns and splicing mechanisms evolved from the
Group II introns.

alternative splicing.
Alternative splicing refers to the process by
which a given gene is spliced into more than
one type of mRNA molecule.
Copyright National Institutes of Health

Early in the course of splicing research, yet another surprising discovery was made; specifically,
researchers noticed that not only was pre-mRNA punctuated by introns that needed to be excised, but also
that alternative patterns of splicing within a single pre-mRNA molecule could yield different functional
mRNAs (Figure 2; Berget et al. 1977). The first example of alternative splicing was defined in the
adenovirus in 1977 and demonstrated that one pre-mRNA molecule could be spliced at different junctions
to result in a variety of mature mRNA molecules, each containing different combinations of exons.
Shortly afterward, alternative splicing was found to occur in cellular genes as well, with the first example
identified in the IgM gene, a member of the immunoglobulin superfamily (Early et al., 1980). Another
example of a gene with an impressive number of alternative splicing patterns is the Dscam gene from
Drosophila, which is involved in guiding embryonic nerves to their targets during formation of the fly's
nervous system. Examination of the Dscam sequence reveals such a large number of introns that
differential splicing could, in theory, create a staggering 38,000 different mRNAs. This ability to create so
many mRNAs may provide the diversity necessary for forming a complex structure such as the nervous
system (Schmucker et al., 2000). In fact, the existence of multiple mRNA transcripts within single genes
may account for the complexity of some organisms, such as humans, that have relatively few genes
(approximately 20,000). For example, work from Wang et al. (2008) suggests that more than 90% of human
genes are alternatively spliced.

The Past and Future of Introns

The existence of introns and differential splicing helps explain how new genes are created during
evolution. Splicing makes genes more "modular," allowing new combinations of exons to be created during
evolution. Furthermore, new exons can be inserted into old introns, creating new proteins without
disrupting the function of the old gene.
Our knowledge of RNA splicing is quite new. Nonetheless, because nearly all eukaryotes have introns and
share mechanisms of RNA splicing, splicing itself must be quite ancient. Proponents of the "intron-early"
theory suggest that all organisms (including prokaryotes) at one time had introns in their genome but
subsequently lost these elements, while "intron-late" supporters believe that the restriction of introns to
eukaryotes suggests a more recent introduction (Roy & Gilbert, 2006). There is no apparent pattern in
which eukaryotes have introns, and that makes it difficult for researchers to make predictions about how
introns were gained or lost through evolution. What is clear, however, is that introns and splicing have
clearly played a significant role in evolution, and scientists are only beginning to discover the nature of that
role.

References and Recommended Reading

Berget, S. M., et al. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proceedings of the National Academy of Sciences 74, 31713175
(1977)
Chow, L. T., et al. An amazing sequence arrangement at the 5 ends of adenovirus 2 messenger RNA. Cell 12, 18 (1977)
Darnell, J. E., Jr. Implications of RNARNA splicing in evolution of eukaryotic cells. Science 202, 12571260 (1978) doi:10.1126/science.364651
Early, P., et al. Two mRNAs can be produced from a single immunoglobulin chain by alternative RNA processing pathways. Cell 20, 313319 (1980)

Knapp, G., et al. Transcription and processing of intervening sequences in yeast tRNA genes. Cell 14, 221236 (1978)
Konarska, M. M., et al. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature 313, 552557 (1984) doi:
10.1038/313552a0 (link

to article)

Patel, A. A., & Steitz, J. A. Splicing double: Insights from the second spliceosome. Nature 4, 960970 (2003) doi:10.1038/nrm1259 (link

to

article)
Pierce, B. A. Genetics: A Conceptual Approach, 2nd ed. (New York, Freeman, 2000)
Roy, S. W., & Gilbert, W. The evolution of spliceosomal introns: Patterns, puzzles, and progress. Nature Reviews Genetics 7, 211221 (2006) doi:
10.1038/nrg1807 (link

to article)

Schmucker, D., et al. Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101, 671684 (2000)