Different alternative splicing mechanisms and there
significance to proteome diversity
Temesgen Dagnaw1
Abstract
In contrast to bacterial and archaeal genes, the vast majority of genes in higher, multicellular
eukaryotes contain multiple introns. Introns present in pre-mRNA derived from the same gene
are alternatively spliced in more than one way, thereby yielding different collections of exons in
the mature mRNA. Such alternative splicing yields a group of similar but nonidentical mRNAs
that, upon translation, result in a series of related proteins called isoforms, which greatly expands
proteomic diversity in higher eukaryotes. Splice site selection is the basic process of splicing to
assemble spliceosome and cut at the place, which gives rise to different types of alternative
splicing in turn increases the diversity of proteome. In this review I discuss seven types of
alternative splicing namely: cassette exon (exon skipping), intron retention, mutually exclusive
exons alternative 5’ and 3’ spice sites selections, alternative promoters, alternative polyadenyaltion sites.
Contents
1) Introduction
2) Molecular Mechanisms of Alternative Splicing
3) Types of Alternative Spicing Events
A. Cassette exon (exon skipping)
B. Intron retention
C. Mutually exclusive exons
D. Alternative 5’ and 3’ spice sites selections
E. Alternative promoters
F. Alternative poly-adenyaltion sites
4) Conclusion
5) Reference
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1) Introduction
Cells have to make a lot of proteins in order to function right. For organisms with more complex
cells, they need to have slightly different proteins to perform similar functions in different
organelles. In multicellular organisms, different proteins with the same function have to be made
for different organs in the body. Instead of having dozens of genes to make separate proteins for
each location, cells will use alternative gene splicing in order to generate different forms of the
protein from one gene.
Alternative splicing is the process in which the primary transcript of a gene is reorganized to
produce a different protein than the primary transcript. By manipulating of the exons, the
sequence of the amino acids produced from the mRNA is affected, resulting in a different protein
sequence, and protein structure. Alternative splicing of pre-mRNA was introduced in 1980 for
the first time when it was discovered that membrane bound and secreted antibodies encoded by
the same gene. Then after many works described this phenomenon, results were considered and
led to the idea that genes can encode more than one protein or protein isoform as a result of
alternative splicing of pre-mRNA.
Many proteins from higher eukaryotes have a multidomain tertiary structure. Individual repeated
protein domains often are encoded by one exon or a small number of exons that code for
identical or nearly identical amino acid sequences. Such repeated exons are thought to have
evolved by the accidental multiple duplication of a length of DNA lying between two sites in
adjacent introns, resulting in insertion of a string of repeated exons, separated by introns,
between the original two introns.
The number of proteins in the proteome is far from the number of genes in different organisms.
For example in drosophila Drosophila melanogaster, a gene code for Down syndrome cell
adhesion molecule (Dscam) can generate ~38,000 distinct mRNA isoforms, a number far in
excess of the total number of genes (~14,500) in the organism. In human 95–100% of premRNAs that contain sequence corresponding to more than one exon are processed to yield
multiple mRNAs.
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Human gene on average contains a mean of 8.8 exons, with a mean size of 145nt. The mean
intron length is 3365nt, and the 5’ and 3’ UTR are 770 and 300 nt, respectively. As a result, a
standard Q gene spans about 27 kbp. After pre-mRNA processing, the average mRNA exported
into the cytosol consists of 1340 nt coding sequence, 1070 nt untranslated regions and a poly (A)
tail (Lander et al., 2001). This shows that more than 90% of the pre-mRNA is removed as introns
and only about 10% of the average premRNA are joined as exonic sequences by pre-mRNA
splicing which seems very small in number to support the complex of human physiological
needs. Human cells are not only capable of accurately recognizing the small exons within the
larger intron context, but are also able to recognize exons alternatively. Alternative pre-mRNA
splicing is a prevalent post-transcriptional gene regulation mechanism.
To consider alternative splicing in a single chromosome level of human species the work done
on chromosome 22 and 19 by Lander and his colleagues found 642 transcripts, covering 245
genes from chromosome 22 i.e. average of 2.6 distinct transcripts per gene and. 1,859 transcripts,
corresponding to 544 genes from chromosome 19 i.e. average 3.2 distinct transcripts per gene.
Protein diversity can be produced by other processes such as the use of alternative transcription
start sites, alternative poly adenylation, RNA editing and post-translational modification. The
contribution of these and other mechanisms to protein diversity is not clear, but alternative
splicing has shown visible proteomic diversity in multicellular eukaryotes.
Thus, alternative splicing has a role in almost every aspect of protein function, including binding
between proteins and ligands, nucleic acids or membranes, localization and enzymatic properties.
Taken together, alternative splicing is a central element in gene expression (Kelemen O, 2013)
Here I present a review on the molecular mechanisms of alternative splicing and different types
that are known on alternative splicing and there importance on proteome diversity is highlighted
and discussed.
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2) Molecular Mechanisms of Alternative Splicing
Introns are demarcated by invariant consensus sequences at their 5'and 3' boundaries. The
pathway of splicing comprises cleavage at the 5’ site, formation of the lariat branchpoint, and
cleavage at the 3’ site with concomitant ligation of the 5' and 3' exons. The central problem in
pre-mRNA splicing, both constitutive and alternative is the selection of the correct pairs of 5’
and 3’ sites to be joined.
Two major steps constitute the basic process of splicing: Assembly of the spliceosome followed
by the actual splicing of pre-mRNA. The spliceosome is mainly composed of U1, U2 small
nuclear ribonucleic proteins (snRNPs) and the U4/U6.U5 tri-snRNP, and configure in identify a
core set of splicing signals: The 5' splice site, the branch point sequence and the 3' splice site
(Fig. 2). Specific spliceosomal complexes (E, A, B and others) and eight evolutionarily
conserved DExD/H-type RNA-dependent ATPases/helicases assemble in a proposed stepwise
manner and execute multiple splicing steps that result in exon ligation and intron excision.
The exons that end up in the mature mRNA during the process of alternative splicing is entirely
defined by the interaction between cis-acting elements and trans-acting factors. Cis-acting
elements include exonic splicing enhancers (ESEs) and intronic splicing enhancers (ISE) that are
bound by positive trans-acting factors, such as SR proteins (serine/arginine-rich family of
nuclear phosphoproteins), whereas exonic splicing silencers (ESSs) and intronic splicing
silencers are bound by negative acting factors, such as heterogeneous nuclear ribonucleoproteins
(hnRNPs). For example, hnRNP M generates alternatively spliced dopamine receptor
pre-mRNAs, which create isoforms associated with diverse key physical functions, such as
control, reward, learning and memory (Park E, Iaccarino C, Lee J, et al., 2011). The
collaboration between these elements results in the promotion or inhibition of splicesome
assembly of the weak splice sites, respectively. The enhancing elements roles in constitutive
splicing, while the silencers role in the control of alternative splicing. On YAN WANG et al.,
(2015) ESE was found to act as an ISE depending on its location in an exon or intron.
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SR proteins interact with U1 snRNP and the 35 kDa subunit of the heterodimeric factor, U2AF
(figure 1). The second subunit of U2AF, U2AF65, binds SF1 and the pyrimidine tract
simultaneously, on the basis of the arginine/serine (RS) rich domain, which results in recognition
and stability of the branch point, as well as polypyrimidine tract sequences.
In general, positive or negative splice-site recognition is regulated through various mechanisms,
such as the local concentration or activity of splicing regulatory factors, under diverse
physiological or pathological conditions
Intron positions were confirmed by applying a stringent criterion that EST or mRNA sequence
shows an exact match of 8 bp in the flanking exonic sequence on each side. Of 53,295 confirmed
introns, 98.12% use the canonical dinucleotides GT at the 59 splice site and AG at the 39 site
(GT-AG pattern). Another 0.76% uses the related GC-AG. About 0.10% used AT-AC, which is
a rare alternative pattern primarily recognized by the variant U12 splicing machinery. The
remaining 1% belongs to 177 types, some of which undoubtedly reflect sequencing or alignment
errors.
Figure 1 Spliceosome complex on on pre-mRNA
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3) Types of Alternative Spicing Events
Data from ESTs and microarray have revealed seven main types of alternative splicing (Blencowe
BJ, 2006) Alternative splicing events can be classified into cassette exon, mutually exclusive
exons, retained intron, alternative 5’ splice sites, alternative 3’ splice sites, alternative
promoters, and alternative poly-A sites.
Figure 2 Types of alternative splicing that are responsible for the generation of functionally
distinct transcripts.
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A. Cassette-type alternative exon (exon skipping)
Cassette exons are short in length, shows abundance of terminal codons, and weak splice signal
which hinder the ability of the splicing mechanism to recognize these exons, and the result is
exon skipping. The splicing of each cassette exon would appear to be independent of that of
others in the gene. When such an exon is retained, the splicing pattern resembles that for a
constitutive gene in which all potential coding sequences are incorporated into the mature RNA
(Figure 2). When it is removed, it is presumably carried on a long intron that also contains its
flanking noncoding sequences. Such alternatively spliced exons represent discrete cassettes of
genetic information encoding peptide subsegments that are differentially incorporated into the
mature gene product. Several genes contain more than one such cassette. If n is the number of
exons in a gene that may each be individually included or excluded in a combinatorial fashion,
then there is a potential for up to 2" different mRNAs to be encoded by the single gene.
It is the most prevalent pattern (~30%) of alternative splicing in vertebrates and invertebrates. As
an example fast skeletal troponin T (TnT) gene have five consecutive cassettes which are spliced
to yield as many as 32 (25) different sequences in the corresponding domain of the protein,
subject to tissue specific and developmental stage-specific regulation (Breitbart, R. E., et al.,
1985). Another example of combinatorial splicing is found in the gene for the myelin basic
protein, which contains two nonconsecutive cassettes, each of which is differentially
incorporated, generating four (22) isoforms (de Ferra, F. et al., 1985)
B. Intron retention
Several other genes incorporate intron sequence into mRNA by failing to splice both members of
a donor-acceptor pair altogether (Figure2). The retained intron necessarily maintains an intact
translational reading frame and creates a longer fusion exon.
Intron retention has been revealed in lower metazoans, (Kim E, Magen A and Ast G, 2007) and
intron retention in human transcripts is positioned primarily in the untranslated regions (UTRs)
(Galante PA et al., 2004) and has been associated with weaker splice sites, short intron length
and the regulation of cis-regulatory elements (Sakabe NJ and de Souza SJ, 2007). Alternative
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splicing of the rat r-fibrinogen gene transcripts retain the complete seventh intron in 10% of the
mRNAs, which add an extra protein isoforms (Crabtree, G. R . et al., 1982).
C. Mutually exclusive exons
Several contractile protein genes contain pairs of consecutive cassette exons that are
differentially spliced in a mutually exclusive fashion. Here, one member or the other of the pair
is invariably spliced into a given mRNA, but the exclusion or inclusion of both simultaneously
does not occur. Each mutually exclusive cassette encodes an alternative version of the same
protein domain in two distinct mRNAs.
Where the pair of mutually exclusive exons is located between a common donor and a common
acceptor, the sequence between them is a pseudointron; although this pseudointron contains
appropriate and functional 5' and 3' splice sites, as evidenced by their capacity to pair with more
distant junctions, it is never excised as a precise unit. This ensures that the joining of one exon of
the pair to the common donor is not followed by the joining of the other to the common acceptor.
D. Alternative 5’ and 3’ spice sites selections
Heterogeneous sites of transcription initiation and of 3' end formation necessarily result in
transcripts having decidedly distinct primary structures. Different promoters and different
polyadenylation sites may specify alternative 5' and 3' terminal exons, respectively. These exons
are not cassettes in the sense defined above, in that each is flanked by a single splice site at its
internal boundary alone. In some instances, alternative splice site usage is also manifest in exons
internal to these heterogeneous termini.
E. Alternative promoters
Two promoters in the mouse a-amylase gene, one active in salivary gland and the other in liver,
initiate alternative first exons (Figure 2). The splicing of the shorter (liver) transcript, is
essentially constitutive because the upstream exon 1 donor site is absent from the RNA and,
hence, not available to the exon 3 acceptor. In the longer (salivary) transcript, the exon 2 donor
site is present but remains unspliced.
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The MLC1I3 gene is one of the best documented examples of mutually exclusive splicing
regulated through the alternative use of two different promoters. Vertebrate fast skeletal muscle
contains two alkali MLC light chain isoforms (MLCI and MLC3) that differ from each other at
the amino-terminus (exons 1 and 2), and where they have additional isoformspecific internal
sequences (exons 4 and 3, respectively).
The gene for the human interleukin-2 receptor has two promoters that are quite closely spaced
and initiate overlapping first exons. Moreover, the fourth exon is a true cassette, encoding an
internal protein domain, which is differentially incorporated (Leonard, W. J. et al., 1984).
F. Alternative poly-adenyaltion sites
The protein expression is further regulated by alternative polyadenylation of mRNA, which
influences the coding potential or the 3'UTR length by modifying the binding availability of
microRNA or RNA. For example, 50% or more of human genes encode multiple transcripts
derived from APA (Tian et al., 2005). Dafne C. G., Kensei N. and James M. have considered two
general classes of APA. In some cases the alternative poly(A) sites are located in internal
introns/exons and therefore APA events produced different protein isoforms, referred to this type
as CR-APA (Coding Region-APA). In other cases, APA sites are all located in the 3’
untranslated region (3’UTR), resulted in transcripts with 3’UTRs of different length but encoded
the same protein; referred to this type of APA as UTR-APA.
While CR-APA can affect gene expression qualitatively by producing distinct protein isoforms
while UTR-APA affect expression quantitatively. 3’UTRs often harbor microRNA (miRNA)
binding sites and/or other regulatory sequences. Longer 3’UTRs will more likely possess such
signals, or more of them, and the mRNA will therefore likely be more prone to negative
regulation. Indeed, the amount of protein generated by an mRNA has been showed to depend on
its 3’UTR length, such that transcripts with shorter 3’UTRs produce higher levels of protein
(Mayr and Bartel, 2009). Furthermore, the length of the 3’UTR can affect not only the stability
but also the localization, transport and translational properties of the mRNA. Differential
processing at multiple poly(A) sites can be influenced by physiological conditions such as cell
growth, differentiation and development, or by pathological events such as cancer.
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Many genes have multiple alternative splicing events with complex combinations of exons,
producing a family of diverse transcript isoforms. For example, in Drosophila melanogaster,
gene Dscam can potentially produce 38,016 different mature mRNAs by different combinations
of 95 cassette exons (Graveley, B. et al., 2004).
4) Conclusion
Alternative splicing has a significant role in expanding proteome diversity. In summary, the
mode of splice site selection and appropriate spliceosome complex formation has played a great
role in deciding the destination of the mRNA and in making protein isoform. The above
mentioned seven patterns of alternative spicing explain how exons come together and joined
together to give rise different mature mRNAs although some of ligation revealed intron
retention. Many genes use combinations of this patterns which expected to give much diverse
mature mRNA isoforms.
5) References
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Breitbart, E., Nguyen, H. T., Medford, R. M., Destree, A. T., Mahdavi, et al. 1985. Cell 41:67-82
Crabtree, G. R., Kant, J. A. 1982. Cell 31: 159-66
Dafne Campigli Di Giammartino, Kensei Nishida, and James L. Manley: Mechanisms and
consequences of alternative polyadenylation. Molecular Cell 43(6): 853–866, 2011.
De Ferra, F., Engh, H., Hudson, L., Kamholz, J., Puckett, C. , et al. 1985. Cell 43:72 1-27.
Galante PA, Sakabe NJ, Kirschbaum-Slager N and de Souza SJ: Detection and evaluation of
intron retention events in the human transcriptome. RNA 10: 757-765, 2004.
Graveley and Brenton R.: Mutually Exclusive Splicing of the Insect Dscam Pre-mRNA Directed
by Competing Intronic RNA Secondary Structures. Cell 123(1): 65–73, 2005.
Kelemen O, Convertini P, Zhang Z, et al: Function of alternative splicing. Gene 514: 1‑30, 2013.
Kim E, Magen A and Ast G: Different levels of alternative splicing among eukaryotes. Nucleic
Acids Res 35: 125-131, 2007.
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Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al., Initial sequencing
and analysis of the human genome. Nature 412(6846):565, 2001.
Leonard, W. J., Depper, J. M., Crabtree, G. R., Rudikoff, S., Pumphrey, J: Molecular cloning and
expression of cDNAs for the human interleukin-2 receptor. Nature 311: 626-31, 1984.
Mayr C, Bartel DP. Widespread shortening of 3'UTRs by alternative cleavage and
polyadenylation activates oncogenes in cancer cells. Cell 138:673–684, 2009.
Nilsen TW and Graveley BR: Expansion of the eukaryotic proteome by alternative splicing.
Nature 463: 457-463, 2010.
Park E, Iaccarino C, Lee J, et al: Regulatory roles of heterogeneous nuclear ribonucleoprotein M
and Nova-1 protein in alternative splicing of dopamine D2 receptor pre-mRNA. J Biol Chem
286: 25301-25308, 2011.
Sakabe NJ and de Souza SJ: Sequence features responsible for intron retention in human. BMC
Genomics 8: 59, 2007.
Tian B, Hu J, Zhang H and Lutz CS: A large-scale analysis of mRNA polyadenylation of human
and mouse genes. Nucleic Acids Res. 33:201–212, 2005.
Yan Wang, Jing Liu, Bo Huang, Yan-Mei Xu, Jing Li, Lin-Feng Huang, Jin Lin, Jing Zhang,
Qing-Hua Min, Wei-Ming Yang and Xiao-Zhong Wang: Mechanism of alternative splicing and
its regulation (Review). Biomedical Reports 3: 152-158, 2015.
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