Alternate splicing Alternative splicing (or differential splicing) is a process by which the exons of the RNA produced by transcription

of a gene are reconnected in multiple ways during RNA splicing. The resulting different mRNAs may be translated into different protein isoforms; thus a single gene may code for multiple protein. Alternative splicing occurs as a normal phenomenon in eu!aryotes where it greatly increases the diversity of proteins that can be encoded by the genome; in humans over "#$ of genes are alternatively spliced. There are numerous modes of alternative splicing observed of which the most common is exon s!ipping. %n this mode a particular exon may be included in mRNAs under some conditions or in particular tissues and omitted from the mRNA in others. Abnormal variations in splicing are also implicated in disease; a large proportion of human genetic disorders result from splicing variants. Abnormal splicing variants are also thought to contribute to the development of cancer.

&ig. Alternative splicing produces two protein isoforms.

&ig. 'emonstration of alternative splicing( %n 'NA the genetic information that includes the code for ma!ing a protein is located in fragments (exons red boxes) which are interrupted by non)coding fragments (introns green boxes). *y the process of alternative splicing the introns are removed and the exons spliced together in different combinations generating different messenger RNAs (mRNA) that are decoded (translated) into distinct proteins. Basic modes of Alternative splicing: &ive basic modes of alternative splicing are generally recogni+ed. %. Exon skipping or cassette exon( in this case an exon may be spliced out of the primary transcript or retained. This is the most common mode in mammalian pre)mRNAs. %%. Mutually exclusive exons( ,ne of two exons is retained in mRNAs after splicing but not both. %%%. Alternative donor site: An alternative -. splice /unction (donor site) is used changing the 0. boundary of the upstream exon. %1. Alternative acceptor site( An alternative 0. splice /unction (acceptor site) is used changing the -. boundary of the downstream exon. 1

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Intron retention( A se2uence may be spliced out as an intron or simply retained. This is distinguished from exon s!ipping because the retained se2uence is not flan!ed by introns. %f the retained intron is in the coding region the intron must encode amino acids in frame with the neighboring exons or a stop codon or a shift in the reading frame will cause the protein to be non)functional. This is the rarest mode in mammals.

&ig. Traditional classification of basic types of alternative RNA splicing events. General splicing mechanism 3hen the pre)mRNA has been transcribed from the 'NA it includes several introns and exons. &or examples in nematodes the mean is 4)- exons and introns; in the fruit fly 'rosophila there can be more than 5## introns and exons in one transcribed pre)mRNA. The exons to be retained in the mRNA are determined during the splicing process. The regulation and selection of splice sites are done by trans)acting splicing activator and splicing repressor proteins. The typical eu!aryotic nuclear intron has consensus se2uences defining important regions. 6ach intron has 78 at its -. end. Near the 0. end there is a branch site. The nucleotide at the branch point is always an A; the consensus around this se2uence varies somewhat. %n humans the branch consensus is y8nAy. The branch site is followed by a series of pyrimidines or polypyrimidine tract then by A7 at 0. end. 9plicing of mRNA is performed by an RNA and protein complex !nown as the spliceosome containing snRN:s (small nuclear ribonucleoprotein particles complexes of snRNAs and proteins) designated 85 8; 84 8- and 8< (80 is not involved in mRNA splicing). 85 binds to -. 78 and 8; binds to branch site (A) with the assistance of the 8;A& protein factors. The complex at this stage is !nown as the spliceosome A complex. &ormation of the A complex is usually the !ey step in determining the ends of the intron to be spliced out and defining the ends of the exon to be retained. The 84 8- 8< complex binds and 8< replaces the 85 position. 85 and 84 leave. The remaining complex then performs two transesterification reactions. %n the first transesterification -. end of the intron is cleaved from the upstream exon and /oined to the branch site A by a ;. -.)phosphodiester lin!age. %n the second transesterification the 0. end of the intron is cleaved from the downstream exon and the two exons are /oined by a phosphodiester bond. The intron is then released in lariat form and degraded.

Fig. A general splicing mechanism 2

Biological significant of Altenative splicing A!" Alternative splicing has long been regarded as a rather rare event in eu!aryotic genomes. Recent analyses of vast amounts of transcript data in human and other organisms suggest that alternative splicing is widespread in mammalian genomes. %n humans it is estimated that alternative splicing occurs in more than <#$ of genes. Alternative splicing is a powerful means of enhancing protein diversity. %t is estimated that over =#$ of all genes are alternatively spliced as a means for producing functionally diverse proteins from a single gene. %n fact the ma/ority of meta+oan genes encode pre) mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Alternative splicing is found extensively in all higher eu!aryotes ,ne of the most dramatic examples of alternative splicing is the Dscam gene in Drosophilia. This single gene contains some 55< exons of which 5= are retained in the final mRNA. 9ome exons are always included; others are selected from an array. Theoretically this system is able to produce 0" #5< different proteins. And in fact over 5" ### different ones have been found in Drosophila. The 'scam proteins are involved in guiding neurons to their proper destination and probably in recognition and phagocytosis of invading bacteria. 3hether a particular segment of RNA will be retained as an exon or excised as an intron depends on the circumstances.
#egulating gene expression

Alternative splicing plays an important role in regulating gene expression. %n the last decade it has been shown that alternative splicing determines binding properties intracellular locali+ation en+ymatic activity protein stability and posttranslational modifications of a large number of proteins. >any proteins are comprised of several domains or modules that serve a particular function. &or example one domain may help the protein bind to another protein while another domain gives the protein en+ymatic activity. %n the genome domains correspond to exons. *y alternative splicing exons i.e. protein domains can be mixed and matched altering the nature of the protein. *y regulating which splice patterns occur in which tissue an organism fine)tunes the action of a single gene so that it can perform many different roles. The conse2uences of alternative splicing fall into two ma/or categories( protein)level alterations and transcript)level modifications. ,n the protein level alternative splicing generates splice variants that give rise to different protein products. &or example a shortened protein product due to a frame shift introduced by an alternate exon or a protein product with a different functional domain due to the inclusion of a specific exon from a mutually exclusive group of exons. ,n the transcript level alternative splicing produces splice variants that have different translation or stability profiles. &or example a transcript with a longer lifespan would prolong the availability of the corresponding protein. Thus it plays an extremely important role in expanding protein diversity and might therefore partially explain the apparent discrepancy between gene number and organism complexity.