discovery of the spliceosomal introns in eukaryotic genes

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The 1977 discovery of the spliceosomal introns in eukaryotic genes, and the subsequent description of corresponding splicing mechanisms that put exons together, were among the most fundamental, amazing, and puzzling discoveries in biology (Chow, Gelinas, Broker, & Roberts, 1977; Berget, Moore, & Sharp, 1977). Spliceosomal introns (group III) are sequences that interrupt nuclear coding sequences in eukaryotes, and are removed from RNA transcripts by a complicated protein-RNA complex, called the spliceosome. The American biochemist Walter Gilbert in 1978 coined the term introns and exons, while he was predicting the role of introns in the gene evolution. According to Walter Gilbert (1978), introns play a key role in exon shuffling and alternative splicing. He states that "the notion of the cistron 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". Introns occupy a large proportion of the non-coding genomic portion of the DNA. Because of transcription of genomic DNA, a precursor mRNA, or pre-mRNA, is generated. The pre-mRNA comprises of four major parts that include 5' untranslated region or 5'-UTR, the exon that is protein coding, the intron that is non-coding and the 3' untranslated region or 3'-UTR. The 5'- and 3'-UTR's are the extensions of the introns; however, the processing of these 5'- and 3'-UTR's differs from the intron that is located between the two exons that are protein coding and this intron is referred to as the in-frame intron. The in-frame intron is lengthy and was considered previously as a genetic waste in the gene transcription (Lin, Miller, & Ying, 2006). The use of introns lays with the fact that distribution of intron lengths in the efforts for large scale sequencing (Irimia & Roy, 2008). The sequences of the spliceosomal introns are quasi-random and in general, they do not contain open reading frames (ORFs) (Roy & Gilbert, 2006). On the contrary, group I and group II introns from a variety of sources have long ORFs which encode proteins to facilitate the propagation of introns to the sites where there are no introns by the process of reverse transcription. Spliceosomes are the macromolecular enzymes that remove the introns from the primary transcripts. Spliceosomes can remove all introns from a primary transcript resulting in the formation of mRNA that is fully spliced or various mRNA's from a gene (Garcia-Blanco, 2003). The spliceosomes are a complex of five RNAs, and the length of the spliceosomes varies with the species. The process of removing the spliceosomal introns is associated with several steps of the transcription processes. Even though there are considerable differences between the spliceosomal introns and the other introns, some similarities exist indicating the possibility of an evolutionary relationship. The number of spliceosomal introns varies greatly in the eukaryotic species. The causes and the timing of evolution of the spliceosomes are interesting in the genomic evolution. Less is known about the mechanisms for the loss or gain of introns, and researches were able to trace the origins for few introns (Roy & Gilbert, 2006). Introns were considered selfish DNA with no specific cell function; however, several different functions of introns in the cell were surveyed. With the recent discoveries of non-coding RNAs with a variety of functions, it can be assumed that there is a possibility of the existence of some functional introns. A case in point is that intronic sequences of the mouse hsc70 heat shock gene are the source of U14 small nuclear RNA, or snRNA. Introns can be regarded as the functional genomic carriers of the gene regulatory elements. Trans-splicing is the process in which two different mRNAs recombine to form a single mRNA, and this process occurs through recombination within the non-coding parts of the molecules that are very similar to introns (Fedorova & Fedorov, 2003). Even though it is not clear about the prevalence of trans-splicing in eukaryotes, it increases the protein diversity. It has been observed that in the chromosomal regions where there is less frequency for meiotic crossing over, introns are supposed to be longer and thus they increase the recombination rate between the exons (Comeron & Kreitman, 2000). Introns are providers for the exon shuffling and this process is extremely important in the evolutionary process. The introns have an intricate interaction of splicing with the processes of export of mRNA from the nucleus and the regulation of the stability of the mRNA in the cytoplasm (Fedorova & Fedorov, 2003). Introns mediate gene regulation via alternative splicing, and these regulatory roles and for this activity, long conserved sequences are not required. This makes introns to evolve freely in a faster way than exons, and a feature of the introns that makes them important tool for the evolutionary studies. This also provides the basis for the development of DNA molecular markers for polymorphism. The splicing in plants differs from animals in recognizing the site of splicing. Plants bear shorter introns and the base composition of introns is strongly different from exons. The splicing requirements for the dicots differ from monocots of plants, and working on plant species is important in that it gives a detailed view on the complexity in plants. In plants, introns regulate the level and the pattern of the expression of the gene during the growth and the morphogenesis under various environmental conditions (Morello & Breviario, 2008). Lastly, the mechanisms by which introns are inserted and deleted from gene loci are not well understood. Intron density differs greatly among organisms, and the evolutionary history of spliceosomal introns remains one of the most hotly debated topics in eukaryotic evolution (Roy & Gilbert, 2006).

Origin of introns

Several questions were raised with regard to the function and the origin of introns after the discovery of these sequences. According to Gilbert (1978), introns had their existence in the ancestral genes and their utility lies in assembling the first genes. Even though there is some evidence for this hypothesis such as the relationship between the exon structure and the domain structure of the proteins, the argument has no theoretical basis (Demetrius, 1988). There might be a split in the structure of the ancestral genes and the relics from the primordial assembly of genes that might have been preserved in the guise of the exons through recombination in introns (Doolittle, 1978). The origin of the spliceosomal introns is the longest unsolved mysteries of molecular biology. After an extensive study for several years, researchers were able to find out the origin of introns in only two cases that include a short interspersed nucleotide element or SINE insertion that resulted in a new intron in the coding region of the catalase gene CatA of rice. In this case, the intron was derived from the ancestral genome of rice after the evolutionary divergence from the other ancestral cereals such as wheat, barley and oat (Iwamoto, Maekawwa, Saito, Higo, & Higo K, 1998; Roy, 2004). In the other case where the origin of introns was observed, two midge globin genes have acquired introns via the gene conversion with an intron containing paralog. The intron that was found in the globin gene is interpreted as an evidence for the three-intron and the four-exon structure of the ancient globin gene (Hankeln, Friedl, Ebersberger, Martin, & Schmidt, 1997; Roy, 2004). However, most of the eukaryotes have multiple introns per gene that requires a large number of gains of introns throughout the evolution of the eukaryotes (Roy, 2004). It is an assumption that the differences in the sizes of population and the rate of mutations correlates with the density distributions of introns among different species. The magnified intron abundance in many lineages of organelle genomes, i.e., chloroplasts and mitochondria, of plants is consistent with the idea that the necessary population size and mutation rate requirements for intron proliferation were satisfied only after the origin of multicellular organisms and the associated reduction in effective population size. (Lynch, 2002). It has been observed from the complete genomic sequences of diverse phylogenetic groups that there is an increase in the complexity of genomes from simple prokaryotes to the multicellular eukaryotes, in which there has been an abrupt increase in the number of spliceosomal introns. These modifications are the passive emergences in response to the reduction in the sizes of populations and an increase in the size of the organism (Lynch & Conery, 2003). The evolution of introns comprises of a background neutral component and a selection driven component. The neutral background may correspond to the balanced evolution mode, in which rates of intron gain and loss rates are accordant. In the selective driven component, the evolution of intron is evident by a significant increase in the gain and a significant decrease in the loss, which results in genes that are evolutionarily conserved have a greater density of introns than the genes which evolve at a faster pace (Carmel, Rogozin, Wolf, & Eugene, 2007). Research reports observe that genes with high expression evolve at slower rates, and the level of expression of genes primarily determine the evolution of the rate of both coding and non-coding DNA sequence. The number of translational events experienced by a gene is the determinants of expression and evolutionary rate (Drummond, Raval, & Wilke, 2006). It has been shown that the introns affect the expression of various genes at various levels that include mRNA export, stability, and efficiency of translation (Nott, Meislin, & Moore, 2003). Introns and the spliceosomes for the process of splicing are present in the ancestors. Splicing is a fundamental aspect of all the eukaryotes and may have evolved before the last ancestor of the living eukaryotes. There might be a considerable period between the first eukaryote and the eukaryote ancestor as it is not possible to ascertain on the origin of spliceosome (Collins & Penny, 2005).