Splicing spliceosomes

Splicing is a processing step of the pre-mRNA to become a mature transcript. This involves the excision of intervening noncoding sequences (introns) from coding sequences (exons) by a multiple protein complex, the spliceosome. After splicing the mRNA molecule is ready for translation, since it contains a continuous sequence that encode an entire protein. 

Spliceosome The macromolecular complex responsible for precursor mRNA splicing. The spliceosome consists of at least five small nuclear RNAs (snRNA Ul, U2, U4, U5, and U6) and many proteins. 

Introns are removed from hnRNA by splicing, accomplished by spliceosomes (also known as an snRNP, or snurp), which are complexes of snRNA and protein. The hnRNA molecule is cut at splice sites at the 5 (donor) and 3 (acceptor) ends of the intron. The intron is excised in the form of a lariat structuie and degraded. Neighboring exons are joined together to assemble the coding region of the mature mRNA. 

Mutations in splice sites affect the accuracy of intron removal from hnRNA during posttran-scriptionai processing. As illustrated in Figure 1-4-4, if a splice site is lost through mutation, spliceosomes may ... 

Spliceosome a protein complex that catalyzes the splicing out of introns in pre-mRNA. 

The removal of introns from pre-messenger RNAs in eukaryotes is catalyzed by the spliceosome, which is a large ribonucleoprotein consisting of at least 70 proteins and five small nuclear RNAs (snRNA) [144]. This splicing pathway involves two phosphotransfer reactions. In the first step, the 5 splice site is attacked by a 2 hydroxy group of an adenosine nucleotide within the intron [indicated by A in Fig. 12] that corresponds to the branch point in the lariat intermediate (Fig. 12,middle). In the second step, the 3 -OH group of the free 5 exon attacks the phosphodiester bond between the intron and... 

Fig. 12 The spliceosome splicing reaction. In the first step, the 2 -OH of an adenosine residue that is conserved in the intron attacks the phosphorus at the 5 splice site and generates an intron-3 -exon 2 intermediate and a free 5 exon 1. In the second step, the free 3 -OH of the 5 exon attacks the phosphorus at the 3 splice site to produce ligated exons and an excised intron...

Of the five snRNAs, U2 and U6 interact with the reaction site (the 5 splice site and the branch point) in the first chemical step. These two snRNAs are known to anneal together to form a stable-based paired structure in the absence of proteins and in the presence of ions as shown in Fig. 13, with U2 acting as an inducer molecule that displaces the U4 (that is an antisense molecule that regulates the catalytic function of U6 RNA) from the initially formed U4-U6 duplex. The secondary (or higher ordered) structure of the U2-U6 complex consists of the active site of the spliceosome. Recent data suggests that these two snRNAs function as the catalytic domain of the spliceosome that catalyzes the first step of the splicing reaction [145]. 

Small nuclear RNAs (snRNAs) are involved in the splicing of mRNA precursors (see p.246). They associate with numerous proteins to form spliceosomes. ... 

Immediately after transcription, the hnRNA introns are removed and the exons are linked to form a continuous coding sequence. This process, known as splicing, is supported by complicated RNA-protein complexes in the nucleus, the so-called spliceosomes. The components of these macromolecular machines... 

Figure 11-4. Splicing of a eukaryotic RNA transcript. A hypothetical hnRNA with two exons (EI and E2) and a single, large intron (I) is shown. Splicing can be divided into two main reactions initial attack of ribose near an A residue within the intron on the splice donor followed by attack of the newly available 3 end of exon I (EI) on the 5 end of exon 2 (E2) with coincident release of the intron. Special sequences surround the splice donor and acceptor sites. All steps occur within the spliceosome complex.

Splicing occurs in a large protein-nucleic acid complex, termed the spUceosome. Components of the spliceosome are, apart from the pre-mRNA, a number of proteins and small RNAs, termed the Ul, U2, U4, U5 and U6. The RNAs found in the spUceo-some are bound to specific proteins. The complexes are termed snRNPs (small nuclear ribonucleoprotein). Depending upon the type of RNA bound, there are Ul, U2, U5 and U4/U6 snRNPs. 

Based on the observation of self splicing by the 23S RNA of Tetrahymena, it is assumed that the cleavage and rejoining of the phosphodiester bond is catalyzed by the RNA components of the spliceosome. The proteins of the spliceosome are believed to be important for the recognition of the 5 and 3 splice sites and for the formation of a defined structure in the spliceosome. Thus, the proteins of the spliceosome play a decisive role in the choice of the splice site and the effeciency of splicing. 

The composition of the spliceosome determines the pattern of chosen sphce sites. Several proteins have been identified which act as antagoiusts in the selection of sphce sites. The SF2 protein (and related proteins) belong to the family of SR proteins (SR Ser- and Arg-rich) and supports the use of 5 -sphce sites. Another protein, the hnRNP A1 protein supports the use of 3 -splice sites. 

The exact mechanism by which Rev interferes with the transport and splicing process is largely unknown. One possible scenario is that Rev interacts with components of the spliceosome, which leads to the release of splice factors that allow cytosolic transport without actual splicing. 

Spliceosomal introns generally have the dinucleotide sequence GU and AG at the 5 and 3 ends, respectively, and these sequences mark the sites where splicing occurs. The Ul snRNA contains a sequence complementary to sequences near the 5 splice site of nuclear mRNA introns (Fig. 26-16a), and the Ul snRNP... 

The known catalytic repertoire of ribozymes continues to expand. Some virusoids, small RNAs associated with plant RNA viruses, include a structure that promotes a self-cleavage reaction the hammerhead ribozyme illustrated in Figure 26-25 is in this class, catalyzing the hydrolysis of an internal phosphodiester bond. The splicing reaction that occurs in a spliceosome seems to rely on a catalytic center formed by the U2, U5, and U6 snRNAs (Fig. 26-16). And perhaps most important, an RNA component of ribosomes catalyzes the synthesis of proteins (Chapter 27). 

Removal of introns Maturation of eukaryotic mRNA usually involves the removal of RNA sequences, which do not code for protein (introns, or intervening sequences) from the primary tran script. The remaining coding sequences, the exons, are spliced together to form the mature mRNA. The molecular machine that accomplishes these tasks is known as the spliceosome. [Note A few eukaryotic primary transcripts contain no introns. Others con tain a few introns, whereas some, such as the primary transcripts for the a-chains of collagen, contain more than fifty intervening sequences that must be removed before mature mRNA is ready for translation.]... 

Figure 28-22 Assembly and action of the spliceosomal complex. Four special sequence elements control the process the 5 and 3 splice sites, the branch point (adenosine A), and a polypyrimidine tract. The snRNP particle U1 locates the 5 splice site and U2 the branch point. The tri-snRNP U4 U6 U5 then binds, U6 recognizing the 5 splice site, and U1 and U4 are released. The 2 -OH of the branch point adenosine attacks the phos-phodiester linkage to form a lariet intermediate, which releases the intron in a lariet form in the final step. After Valcarcel and Green.612...

The spliceosome. The hnRNA of nuclei, which includes all of the pre-mRNA, is associated with proteins, which sometimes form very large 200S particles.604 After limited cleavage with nucleases they tend to sediment in the 30S-40S range and to contain a variety of proteins.605 606 Some of the proteins may have been involved in control of transcription.606 Others participate in splicing. The smaller snRNP particles then appear to come into the nucleus and displace much, but not all, of the protein present in the pre-mRNA ribonucleoprotein particles.605... 

Yeast protein L30, which is not homologous to any bacterial protein, controls its own synthesis by a feedback inhibition at the mRNA splicing step. L30 binds to its own pre-mRNA near the 5 splice site, blocking completion of the spliceosome assembly (Chapter 28).159... 

The mechanism of self-splicing in this case is somewhat different from that observed in the spliceosome reaction (fig. 28.21). First the 3 hydroxyl group of the guanosine cofactor attacks the phosphodiester bond at the 5 splice site. This is followed by another transesterification reaction in which the 3 hydroxyl group of the upstream RNA attacks the phosphodiester bond at the 3 splice site, thereby completing the splicing reaction. The final reaction products include the spliced rRNA and the excised oligonucleotide. 

Since its initial discovery, self-splicing has been found to occur for RNAs from a wide variety of organisms. Certain precursor RNAs that exhibit self-splicing produce lariats, just like those seen in the commonly observed splicing reactions that are catalyzed by spliceosomes (see fig. 28.19). These findings suggest that at one time all splicing reactions were RNA-catalyzed. 

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