RNA splicing reactions

The recombination process is not as precise as the site-specific recombination described earlier, so additional variation occurs in the sequence at the V-J junction. This increases the overall variation by a factor of at least 2.5, thus the cells can generate about 2.5 X 1,200 = 3,000 different V-J combinations. The final joining of the V-J combination to the C region is accomplished by an RNA-splicing reaction after transcription, a process described in Chapter 26. 

DNA ligase and RNA ligase catalyze phosphodiester bond formation between 5 -phosphate and the 3 -hydroxyl ends of DNA or RNA. DNA ligases are required for DNA replication and RNA ligases are required for certain RNA splicing reactions. The function and overall reaction mechanism for DNA ligases are reviewed in Volume X of this series (80). The chemical mechanistic pathway for DNA ligase is outlined in reactions (23a)-(23c). 

A detailed 500 MHz H and P nmr study has been undertaken on branched RNA structures that model the lariat formed in RNA splicing reactions. The study was aided by examining the conformation of the ribonucleotides (248 - 252) which preserve the essential structural elements of an admosine branch point while rentoving the intramolecular base stacking interactions.Detailed studies on the decamer (253) showed that the uridine at the 5 -position of the branch point prefers to stack with the 2 -strand of the branch rather than the 3 -... 

FIGURE 14.23 RNA splicing in TetraAjimejta rRNA matnradon (a) the gnanosine-mediated reaction involved in the antocatalytic excision of the Tetrahymena rRNA intron, and (b) the overall splicing process. The cyclized intron is formed via nncleophilic attack of the 3 -OH on the phosphodiester bond that is 15 nncleotides from the 5 -GA end of the spliced-ont intron. Cyclization frees a linear 15-mer with a 5 -GA end. 

Figure 10.13 Phosphoryl-transfer reactions. The figure shows (a) nucleotide polymerization, (b) nucleic acid hydrolysis, (c) first cleavage of an exon-intron junction by group I ribozyme (d) and by a group II ribozyme, (e) strand transfer during transposition and (f) exon ligation during RNA splicing. (From Yang et al., 2006. Copyright 2006, with permission from Elsevier.)...

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]. 

Some components of the splicing apparatus appear to be tethered to the CTD of RNA polymerase II, suggesting an interesting model for the splicing reaction. As the first splice junction is synthesized, it is bound by... 

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). 

RNA Splicing What is the minimum number of transesterification reactions needed to splice an intron from an mRNA transcript Explain. 

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. 

The primary RNA transcript from a protein-coding gene in a eukaryotic cell must be modified by several RNA processing reactions in order to become a functional mRNA molecule. The 5 end is modified to form a 5 cap structure. Most pre-mRNAs are then cleaved near the 3 end and a poly(A) tail is added. Intron sequences are removed by RNA splicing. 

The small nuclear ribonucleoprotein particles (snRNPs or snurps ) that carry out the splicing reaction use RNA-RNA basepairing to select the splice sites. Almost all intron-exon junctions contain the sequence AG-GU with the GU beginning the intron sequence. Furthermore, the consensus sequence for the beginning of the intron has a longer sequence complementary to the U1 RNA. Thus, assembly of the splicing complex, called the spliceosome, starts when the RNA component of the U1 snRNP base pairs with the junction between the 3 end of the exon and the 5 end of the intron. See Figure 12-13. 

One final class of eukaryotic RNA that deserves mention are the small nuclear RNAs (snRNAs). As their name suggests, this class of RNA is found in the nucleus and participates in the splicing of in-trons from certain types of eukaryotic mRNAs. Five snRNAs (Ul, U2, U4, U5, and U6) take part in these splicing reactions when combined with a... 

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