Biochemistry of Medicinals I Phar 6151 CHAPTER FOUR
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor
 
RNA Splicing and Catalysis
 
I.          RNA Editing
 
RNA from eukaryotes is often edited in order to change the information content of the RNA after transcription.  DNA gene sequences do not always correspond to the sequence of the final protein after translation due to changes in the message at the mRNA level.  There are several mechanism to accomplish this depending on ® the species and tissue.
 
A         Cytidine Deaminase
 
Precurser mRNA is deaminated by cytidine deaminase, thus producing .  This results in;
 
            •           A codon change
            •           Protein with different sequence
            •           Apo-lipo-protein B serves as an example
 
              Apo-B   (liver)             
                  ­ Translation
5'————CAA——————3'
              || Cytidine deaminase
5'————AA——————3'
                  || Translation
              Apo-B     (S.I.)             
 
B.                                                                   RNA Splicing
 
DNA sequences of genes from eukaryotes often harbor regions that are not translated, or intron. Introns "intervene" between portions of sequence that is translated or "expressed" referred to as exons. Therefore, the mRNA must be modified in order to remove the introns before translation.  This is accomplished by splicing.
 
1.       Requirements:
 
            •           An central A
            •           5' end needs AGGAAG 
            •           3' end needs (Py)nNCAGG
            •           A series of snRNPs (small nuclear ribo-nucleo-protein particles) referred to as splicesome
 
Splicing Signals
                                                             Intron                                          
   Upstream Exon æ                                     ­                                           ö   Downstream Exon
5' ||||||||||||||||||||||||||AGGAAG————A——————(Py)nNCAGG|||||||||||||||||||||||||||||||| 3'
                 5' Splice ­ Site              Branch ­ Site                               ­ 3' Splice Site
 
2.       Mechanism
     The mechanism of splicing for exon coupling and intron extrusion depends on two 2 trans-esterification reactions, which result in a lariat form or RNA and the product.
                             Upstream Exon
||<--GAAGGA|||||||||||||||||||||||||| 5'
||                          ­
||                         ­  OH
||                   <--¬ |                                                 Downstream Exon
||--————A——————(Py)nNCAGG|||||||||||||||||||||||||||||||| 3'
                             ||                                      Splice ­ 3' Site
                             ||
                             Upstream Exon
||<--GAAG                   GA|||||||||||||||||||||||||| 5'
||                          |                            |
||                         O—PO2          OH
||                                  /                      ||-----------------------||
||                               O                                                        ||
||                                |                                            ||  Downstream Exon
||--————A——————(Py)nNCAGG|||||||||||||||||||||||||||||||| 3'
                             ||                                      Splice ­ 3' Site
                             ||
                            
||<--GAAG
||                          |
||                         O—PO2
||                                  /
||                               O
||                                |                                                                   Upstream Exon        Downstream Exon
||---————A——————(Py)nNCAG—OH         5' ||||||||||||||||||||||||||AGG|||||||||||||||||||||||||||||||| 3'
                 Lariat Intron                                                                                             Spliceed Product of Exons
 
• The branching associated with the A of the lariat form contains two 2 phospho-di-ester linkages
                 to the 2' and 3' of the A.
 
      Structure of Branch
 
3.       Spliceosomes
 
The spliceo-some which catalyzes the splicing reaction is composed of the following species, which are small proteins made up of RNA and protein.
 
            •           Segments of RNA play a key role in directing the alignment splice sites and in catalysis
•ATP-powered proteins unwind RNA duplex intermediates thus causing the release of snRNPs from the products
 
snRNP Role
U1 Binds the 5' splice site and then the 3' splice site
U2 Binds the branch site and forms part of the catalytic center
U5 Binds the 5' splice site
U4 Masks the catalytic activity of U6
U6 Catalyzes splicing
 
II.        RNA Catalysis

 

      Biological catalysis always takes place in the presence of an enzyme or protein that is engineered to catalyze the reaction in question. Recently, this dogma has been challenged since it has been shown that RNA can self splice without the assistance of proteins.  These RNAs are referred to as Ribozymes. They were first 1st isolated from the ciliated protozoan, Tetrahymena.
                                                                                                       A. Characteristics
            •           Need a cofactor; GMP, GDP or GTP
            •           Two 2 trans-esterification reactions occur
            •           Divalent 2 valent cation is required Mg2+ 
            •           Reaction is highly stereospecific
                                                                                     Mechanism of Ribozyme Self Splicing
                                     ||¬¬¬¬¬¬¬¬¬¬¬¬­
                                     ||     ---||                            ­
                                     ||--G ||-----------G     ­
                                             ¯\                       |      ­
5' Upstream Exon                ¯OH                  |      ­
||||||||||||||||||||||||||||CCCA--||                              PØ   ­ Downstream Exon 3'
                               ||<--AGGGAGG<--||                          G|||||||||||||||||||||||||||||||||||
                               ||-----------------------------------------­
||
                                     ||¬¬¬¬¬¬¬¬¬¬¬¬­
                                     ||     --||¯                            ­
                                     ||--­    ||----------G     ­
5' Upstream Exon               3'                        \      ­
||||||||||||||||||||||||||||CCC—OH--------P¬   ­ Downstream Exon 3'
                               ||<AGGGAGGAG 5'                    G|||||||||||||||||||||||||||||||||||
                               ||---------------------------------­
¯
                                     ||¬¬¬¬¬¬¬¬¬¬¬¬­
                                     ||     --||¯                            ­
                                     ||--­    ||---------------G     ­
                                                                   3' |      ­
                                                                   HO     ­
                               ||<AGGGAGGAG 5'                          ­       5' ||||||||||||||||||||||||||||CCCG|||||||||||||||||||||||||||||||||| 3'
                               ||---------------------------------­
                                 Linear Intron                                                                       Spliceed Product of Exons
 
Short sequences which function as ribozymes have been isolated from various plant viruses. The are called hammerhead ribozymes because of their resemblance to the head of a hammer. They will effectively bind an RNA molecule and carry out on cleavage reaction.  In this reaction the two 2 pieces are not joined by an additional trans-esterification.                       Ribozyme
                                             /-----A------\
                                                            G
                                            |                   |
                                           G                
                                            |                   |
                                         /-A                 C-\
                          CGCGG                           AGCCGG-ppp
                          GCGCC                           CGAGC
                                        \-G-A-A-A=-C-/ ¬¬¬¬Cleavage Site
                                                      C=G
                                                   /-A==-\
                                                  C          G
                                                   \----C---/
                               Substrate