The splicing process occurs in cellular machines called spliceosomes, in which the snRNPs are found along with additional proteins. Most splicing occurs between exons on a single RNA transcript, but occasionally trans-splicing occurs, in which exons on different pre-mRNAs are ligated together. These sequences, which help position the splicing apparatus, are found in the exons of genes and bind proteins that help recruit splicing machinery to the correct site. In addition to consensus sequences at their splice sites, eukaryotic genes with long introns also contain exonic splicing enhancers (ESEs). The adjoining exons are covalently bound, and the resulting lariat is released with U2, U5, and U6 bound to it. This step occurs by transesterification in this case, an OH group at the 3′ end of the exon attacks the phosphodiester bond at the 3′ splice site. With the participation of U5, the 3′ end of the intron is brought into proximity, cut, and joined to the 5′ end. Next, the snRNPs U2 and U4/U6 appear to contribute to positioning of the 5′ end and the branch point in proximity. The guanine residue is thus cleaved from the RNA strand and forms a new bond with the adenine. The bonding of the guanine and adenine bases takes place via a chemical reaction known as transesterification, in which a hydroxyl (OH) group on a carbon atom of the adenine "attacks" the bond of the guanine nucleotide at the splice site. The cut end then attaches to the conserved branch point region downstream through pairing of guanine and adenine nucleotides from the 5′ end and the branch point, respectively, to form a looped structure known as a lariat (Figure 1). First, the pre-mRNA is cleaved at the 5′ end of the intron following the attachment of a snRNP called U1 to its complementary sequence within the intron. Splicing occurs in several steps and is catalyzed by small nuclear ribonucleoproteins ( snRNPs, commonly pronounced "snurps"). Rarely, alternate splice site sequences are found that begin with the dinucleotide AU and end with AC these are spliced through a similar mechanism. A typical sequence is YNYYRAY, where Y indicates a pyrimidine, N denotes any nucleotide, R denotes any purine, and A denotes adenine. The branch point always contains an adenine, but it is otherwise loosely conserved. Another important sequence occurs at what is called the branch point, located anywhere from 18 to 40 nucleotides upstream from the 3′ end of an intron. These consensus sequences are known to be critical, because changing one of the conserved nucleotides results in inhibition of splicing. Most commonly, the RNA sequence that is removed begins with the dinucleotide GU at its 5′ end, and ends with AG at its 3′ end. These sites are found at the 5′ and 3′ ends of introns. Introns are removed from primary transcripts by cleavage at conserved sequences called splice sites. The biochemical mechanism by which splicing occurs has been studied in a number of systems and is now fairly well characterized. Other hypotheses proposed that the DNA template in some way looped or assumed a secondary structure that allowed transcription from noncontiguous regions (Darnell, 1978). These observations solidified the hypothesis that splicing of large initial transcripts did, in fact, yield the mature mRNA. Splicing of RNA transcripts was then observed in several in vitro systems derived from eukaryotic cells, including removal of introns from transfer RNA in yeast cell-free extracts (Knapp et al., 1978). Subsequent to the adenoviral discovery, introns were found in many other viral and eukaryotic genes, including those for hemoglobin and immunoglobulin (Darnell, 1978). Studies of early infection revealed long primary RNA transcripts that contained all of the sequences from the late RNAs, as well as what came to be called the intervening sequences (introns). These mosaics were found late in viral infection.
#Splice react series#
These scientists identified a series of RNA molecules that they termed "mosaics," each of which contained sequences from noncontiguous sites in the viral genome (Berget et al., 1977 Chow et al., 1977). However, in 1977, several groups of researchers who were working with adenoviruses that infect and replicate in mammalian cells obtained some surprising results. In other words, there is a one-to-one correspondence of bases between the gene and the mRNA transcribed from the gene (excepting 5′ and 3′ noncoding regions). Most bacterial RNA transcripts do not undergo splicing these transcripts are said to be colinear, with DNA directly encoding them. Gene regulation was first studied most thoroughly in relatively simple bacterial systems.
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