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DNA Splicing: When pre-mRNA has been described from DNA, it includes several introns and exons. The exons to be retained in mRNA are determined during splicing process. The regulation and selection of splice sites are done by trans-acting splicing activator and splicing represser proteins. “Process by which non-coding sequences of base pairs (introns) are subtracted from the coding sequences (exons) of a gene to transcribe DNA in to mRNA”. Very important phenomenon and needs perfection. Abnormal splicing contributes to different genetic disorders which leads to different genetic diseases. Large number of human genetic disorders results from splicing variants. Steps involved in Splicing: 1. Introns loop out as snRNPs (small nuclear ribonucleoprotein particles) snRNPs (snRNAs + Protein) 2. Introns are excised and exons are joined together. 3. The resulting mRNA may then exit nucleus.. DNA →→→→ Complementary RNA / Pre-mature RNA / nRNA →→→→ mRNA Introns are looped out and cut away by snRNPs and exons are joined. Splicing occur in close vicinity of gene and is frequently co-transcriptional. Transcription and splicing are extremely complex multi-molecular process that involve Protein -- DNA, Protein – RNA and protein – protein interactions. Evidences proved that transcriptional elongation and splicing can be influenced reciprocally. Removal of introns and splicing of exons is done by spliceosomes (complex of 5 snRNAs and 145 proteins). Introns mostly start with words GU and ends with AG which guide the spliceosomes. Alternative splicing: “It is a process by which the exons of the RNA produced by transcription of a gene (a primary gene transcript or pre-mRNA) 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 proteins.” A mechanism by which different forms of mature mRNAs are generated from the same gene. Alternative splicing is a regulatory mechanism by which variations in the incorporation of the exons, or coding regions, into mRNA leads to the production of more than one related protein, or isoform. “The processing of pre-mRNA for many proteins proceeds along various paths in different cells or under different conditions.” Mechanism of alternative splicing is highly variable and new examples are constantly being found, particularly through the use of high-throughput techniques. Alternative splicing of pre-mRNA is a prominent mechanism to generate protein diversity, yet its regulation is poorly understood. Alternative splicing was first observed in 1977 (Adenoviruses produced to different primary transcripts). Provides mechanism for producing a wide variety of proteins from a small number of genes. In human turn out of 23 thousands genes and make at least 10 times different proteins. Pattern of alternative splicing differs consistently in different tissues and must be regulated. Alternative splicing not only produce different proteins from single gene but also different 3' UTRs and 5' UTRs. In human over 80% of genes are alternatively spliced. For example: Dscam gene in drosophila; This gene contains 116 exons of which 17 are retained in the final mRNA. Some exons are always selected for final mRNA transcript and others are selected from the array. Theoretically it can produce more than 38016 different proteins and more than 18000 have been found. Alternative Splicing Trans-splicing “In this phenomenon two different transcripts have been spliced together to form final RNA.” Trans-splicing involves the RNA splicing of separate precursors giving rise to one mature mRNA. It is estimated that about half of the roughly 35000 – 45000 human genes are involved in brain differentiation and trans-splicing type of RNA regulation may also be implicated in the similar processes including development and differentiation. For example: Synthesis of single RNA by splicing together transcripts from; 1. Loci located far apart on the same chromosomes. 2. Opposite strands of the same gene locus. 3. Two alleles of the gene on their separate (homologue) chromosome.