resulting in different diseases depending on which gene is affected. examples of splicing aberrations disease type gene symbol mutation location original donor/acceptor mutated donor/acceptor aberration effect colon cancer apc intron 2 aaggtagat aaggaagat skipping of exon 3 colorectal cancer msh2 exon 15 gaggtttgt gaggtttct skipping of exon 15 retinoblastoma rb1 intron 23 tcttaacttgacaga tcttaacgtgacaga new splice acceptor, intron inclusion trophic benign epidermolysis bullosa col17a1 intron 51 agcgtaagt agcataagt lead to exon skipping, intron inclusion, or the use of a cryptic splice site, resulting in either a truncated protein or a protein lacking a small region of the coding sequence choroideremia chm intron 3 caggtaaag cagataaag premature termination codon cowden syndrome pten intron 4 gaggtaggt gagataggt premature termination codon within exon 5 an example of splicing aberration (exon skipping) caused by a mutation in the donor splice site in the exon 8 of mlh1 gene that led to colorectal cancer is given below. this example shows that a mutation in a splice site within a gene can lead to a profound effect in the sequence and structure of the mrna, and the sequence, structure and function of the encoded protein, leading to disease. exon skipping caused by a donor mutation in the gene mlh1 leading to colorectal cancer. the generation of a mrna from a split gene involves the transcription of the gene into the primary rna transcript, and the precise removal of the introns and the joining of the exons from the primary rna transcript. a deleterious mutation within the splicing signals (donor or acceptor splice sites) can affect the recognition of the correct splice junction and lead to an aberration in the joining of the authentic exons. depending on if the mutation occurs within the donor or the acceptor site, and the particular base that is mutated within the splice sequence, the aberration could lead to the skipping of a complete or partial exon, or the inclusion of a partial intron or a cryptic exon in the mrna produced by the splicing process. either of these situations will usually lead to a premature stop codon in the mrna and result in a completely defective protein. the s&s algorithm aids in determining which splice site and exon in a gene are mutated, and the s&s score of the mutated splice site aids in determining the type of splicing aberration and the resulting mrna structure and sequence. the example gene mlh1 affected in colorectal cancer is shown in the figure. it was found using the s&s algorithm that a mutation in the donor splice site in exon 8 led to the skipping of the exon 8. the mrna thus lacks the sequence corresponding to exon 8 (sequence positions are shown in the figure). this causes a frame shift in the mrna coding sequence at amino acid position 226, leading to premature protein truncation at amino acid position 233. this mutated protein is completely defective, which has led to colorectal cancer in the patient. s&s in cryptic splice sites research and medical applications the proper identification of splice sites has to be highly precise as the consensus splice sequences are very short and there are many other sequences similar to the authentic splice sites within gene sequences, which are known as cryptic, non-canonical, or pseudo splice sites. when an authentic or real splice site is mutated, any cryptic splice sites present close to the original real splice site could be erroneously used as authentic site, resulting in an aberrant mrna. the erroneous mrna may include a partial sequence from the neighboring intron or lose a partial exon, which may result in a premature stop codon. the result may be a truncated protein that would have lost its function completely. shapiro–senapathy algorithm can identify the cryptic splice sites, in addition to the authentic splice sites. cryptic sites can often be stronger than the authentic sites, with a higher s&s score. however, due to the lack of an accompanying complementary donor or acceptor site, this cryptic site will not be active or used in a splicing reaction. when a neighboring real site is mutated to become weaker than the cryptic site, then the cryptic site may be used instead of the real site, resulting in a cryptic exon and an aberrant transcript. numerous diseases have been caused by cryptic splice site mutations or usage of cryptic splice sites due to the mutations in authentic splice sites. s&s in animal and plant genomics research s&s has also been used in rna splicing research in many animals and plants. the mrna splicing plays a fundamental role in gene functional regulation. very recently, it has been shown that a to g conversions at splice sites can lead to mrna mis-splicing in arabidopsis. the splicing and exon–intron junction prediction coincided with the gt/ag rule (s&s) in the molecular characterization and evolution of carnivorous sundew (drosera rotundifolia l.) class v b-1,3-glucanase. unspliced (lsdh) and spliced (ssdh) transcripts of nad+ dependent sorbitol dehydroge nase (nadsdh) of strawberry (fragaria ananassa duch., cv. nyoho) were investigated for phytohormonal treatments. ambra1 is a positive regulator of autophagy, a lysosome-mediated degradative process involved both in physiological and pathological conditions. nowadays, this function of ambra1 has been characterized only in mammals and zebrafish. diminution of rbm24a or rbm24b gene products by morpholino knockdown resulted in significant disruption of somite formation in mouse and zebrafish. dr.senapathy algorithm used extensively to study intron-exon organization of fut8 genes. the intron-exon boundaries of sf9 fut8 were in agreement with the consensus sequence for the splicing donor and acceptor sites concluded using s&s. the split-gene theory, introns and splice junctions the motivation for dr. senapathy to develop a method for the detection of splice junctions came from his split-gene theory. if primordial dna sequences had a random nucleotide organization, the random distribution of stop codons would allow only very short open reading frames (orfs), as three stop codons out of 64 codons would result in an average orf of ~60 bases. when senapathy tested this in random dna sequences, not only this was proven to be true, but the longest orfs even in very long dna sequences was found to be ~600 bases above which no orfs existed. if so, a long coding sequence of even 1,200 bases (the average coding sequence length of genes from living organisms), and longer coding sequences of 6,000 bases (many of which occur in living organisms) will not occur in a primordial random sequence. thus, genes had to occur in pieces in a split form, with short coding sequences (orfs) that became exons, interrupted by very long random sequences that became introns. when the eukaryotic dna was tested for orf length distribution, it exactly matched that from random dna, with very short orfs that matched the lengths of exons, and very long introns as predicted, supporting the split gene theory. if this split gene theory was true, then the ends of these orfs that had a stop codon by nature would have become the ends of exons that would occur within introns, and that would define the splice junctions. when this hypothesis was tested, the almost all splice junctions in eukaryotic genes were found to contain stop codons exactly at the ends of introns, bordering the exons. in fact, these stop codons were found to form the “canonical” ag:gt splicing sequence, with the three stop codons occurring as part of the strong consensus signals. the nobel laureate dr. marshall nirenberg, who deciphered the codons, stated that these findings strongly showed that the split gene theory for the origin of introns and the split structure of genes must be valid, and communicated the paper to the pnas. new scientist covered this publication in “a long explanation for introns”. this basic split gene theory led to the hypothesis that the splice junctions originated from the stop codons. besides the codon cag, only tag, which is a stop codon, was found at the ends of introns. surprisingly, all three stop codons (tga, taa and tag) were found after one base (g) at the start of introns. these stop codons are shown in the consensus canonical donor splice junction as ag:gt(a/g)ggt, wherein the taa and tga are the stop codons, and the additional tag is also present at this position. the canonical acceptor splice junction is shown as (c/t)ag:gt, in which tag is the stop codon. these consensus sequence clearly show the presence of the stop codons at the ends of introns bordering the exons in all eukaryotic genes. dr. marshall nirenberg again stated that these observations fully supported the split gene theory for the origin of splice junction sequences from stop codons, who was the referee for this paper. new scientist covered this publication in “exons, introns and evolution”. dr. senapathy wanted to detect the splice junctions in random dna based on the consensus splice signal sequences, as he found that there were many sequences resembling splice sites that were not the real splice sites within genes. this position weight matrix method turned out to be a highly accurate algorithm to detect the real splice sites and the cryptic sites in genes. he also formulated the first exon detection method, based on the requirement for splice junctions at the ends of exons, and the requirement for an open reading frame that would contain the exon. this exon detection method also turned to be highly accurate, detecting most of the exons with few false positives and false negatives. he extended this approach to define a complete split gene in a eukaryotic genomic sequence. thus, the pwm based algorithm turned out to be very sensitive to not only detect the real splice sites and cryptic sites, but also to detect mutated splice sites that are deleterious as opposed to non-deleterious splicing mutations. the stop codons within splice junctions turned out to be the strongest bases in splice junctions of eukaryotic genes, when tested using the pwms of the consensus sequences. in fact, it was shown that mutations in these bases were the cause of diseases compared to other bases, as these three of the four bases (base 1, 3 and 4) of the canonical ag:gt were part of the stop codons. senapathy showed that, when these canonical bases were mutated, the splice site score became weak, causing splicing aberrations in the splicing process and translation of the mrna (as described under the diseases section above). although the value of the splice site detection method in discovering genes with splicing mutations that caused disease has been realized over the years, its importance in clinical medicine is increasingly realized in the next generation sequencing era over the past five years, with its incorporation in several tools based on the s&s algorithm. dr. senapathy is currently the president and cso of genome international corporation (gic), a genomics r&d company based in madison, wi. his team has developed several databases and tools for the analysis of splice junctions, including eusplice, aspalt, exdom and robust. aspalt was commended by biotechniques, which stated that it solved a difficult problem for scientists in the comparative analysis and visualization of alternative splicing across different genomes. gic has most recently developed the clinical genomics analysis platform genome explorer®. selected publications shapiro, marvin b.; senapathy, periannan (1987). "rna splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression". nucleic acids research. 15 (17): 7155–7174. doi:10.1093/nar/15.17.7155. pmc 306199. pmid 3658675. senapathy, p. (1988). "possible evolution of splice-junction signals in eukaryotic genes from stop codons". proc natl acad sci u s a. 85 (4): 1129–33. bibcode:1988pnas...85.1129s. doi:10.1073/pnas.85.4.1129. pmc 279719. pmid 3422483. senapathy, p; shapiro, mb; harris, nl (1990). "splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project". methods in enzymology. 183: 252–78. doi:10.1016/0076-6879(90)83018-5. pmid 2314278. harris, n.l.; senapathy, p. (1990). "distribution and consensus of branch point signals in eukaryotic genes: a computerized statistical analysis". nucleic acids res. 18 (10): 3015–9. doi:10.1093/nar/18.10.3015. pmc 330832. pmid 2349097. senapathy, p. (1986). "origin of eukaryotic introns: a hypothesis, based on codon distribution statistics in genes, and its implications". proc natl acad sci u s a. 83 (7): 2133–7. bibcode:1986pnas...83.2133s. doi:10.1073/pnas.83.7.2133. pmc 323245. pmid 3457379. regulapati, r.; bhasi, a.; singh, c.k.; senapathy, p. (2008). "origination of the split structure of spliceosomal genes from random genetic sequences". plos one. 3 (10): 10. bibcode:2008ploso...3.3456r. doi:10.1371/journal.pone.0003456.