RNA Splicing Part 2 Protein Synthesis : Take Home Message
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor
 
Required reading: Stryer 4th Ed. Ch. 34 p. 888-908
 
RNA Splicing: Take Home Message
 
1) mRNA can be modified by either deamination or splicing.
 
2) Splicing involves the bringing together of exons (expressed sequences) and
          the removing of introns (intervening sequences) in a very specific and defined way.
 
3) RNA can carry out catalytic enzymatic like reactions.
 
Prokaryotic vs. eukaryotic RNA processing 
 
 
Prokaryotes:
 
1) Primary transcript is translated directly
          (no splicing)
 
2) mRNA are often «polycistronic» (i.e. encode more than one 1 polypeptide) 		 
Eukaryotes:
 
1) Primary transcript is processed - capped,
          PolyA added, spliced
 
2) mRNA are transported to cytoplasm
 
3) Each mature mRNA codes for  one 1 polypeptide
 
 
Prokaryotic mRNAs often are polycistronic
(encode more than one polypeptide)
 
(a)  transcription in the presence of an inducer

RNA polymerase binds, transcription of structural genees begins
Inactivated repressor cannot bind to operator
Inducer molecules bind to repressor, inactivating it Beta-Galactosidase   Permease   Acetyl transferase
 
mRNA processing in eukaryotes:
 


                              Nascent RNA ¯              ­ Cleavage signal
                              Nascent RNA ¯¬ Cleavage  by specific
                              Nascent RNA ¯¬              endonuclease 
                                           ATP®¯¬   Addition of tail by
                                            PPi¬ ¯¬  poly(A) polymerase
5' CapAAUAAA  AAAAAAAAAAAAAAAA(A)n
Polyadenylated mRNA precursor
 
Figure 33-32, page 859 Stryer: Biochemistry, Fourth Edition 1995 by W.H.Freeman and Company
 
Termination and RNA splicing
5'®…UCCCAGCCCGCCUAAU      RNA strand
                                                           G                                      unwinded antisence single strand DNA
                                                               A   GCCCG AAAAAAAA   C   T 
                                                                 G/  |  |  |  |   |   |  |  |  |  |  |  |  |                T
                                                                C \ CGGGCUUUUUUUU-OH®3'          GTTTT¬5'
                                                            T RNA polymerase Þ movement Þ                |  |  |  |  |
DNA double strand                        C                                                                            C AAAA ®3'
3'¬GGG T CGGGCGGATTCA                                                                            A A
        |  |   |  |   |  |   |   |  |   |  |  |  |  |  |  |                                   T T T T T T T T G
5'®CCCA GCCCGCC TAAGT G A G C G G G C unwinded sence single strand DNA
 
                                                                ®G®  
                                                              A            U stable hairpin formation
                                                              A          G
                                                                U      A 
                                                                 CºG  ¬     Doublestranded
                                                                 GºC ¬      Spliced tertiary
                                                                 CºG  ¬     3° structure RNA
                                                                 CºG  ¬        hairpin Loop
                                                                 CºG  ¬             formation
                                                                 GºC  ¬                Region
                                        5'®C•C•C•A-/     \ U
                                                                            U A A A  A A A A  C T
                                                                                U / |   |   |    |    |   |                  T
DNA double strand                                                 A \ U U U U U U -OH®3'   G T T T T ¬5'
3'¬GGG T CGGGCGGA TTCA C T CGCCCG                                                  |   |  |   |  |
        |  |   |  |   |  |   |   |  |   |  |  |  |   |  |  |   |   |   |  |  |   |  |  |     T  T  T  T  T T  T  T G AAC AA AA ®3'
5'®CCCA GCCCGCC TAAG T G AGCG GGC unwinded sence single strand DNA
 
                                                                ®G®
                                                              A            U
                                                              A          G
                                                                 U      A
                                                                 CºG  ¬     Doublestranded
                                                                 GºC ¬      Spliced tertiary
                                                                 CºG  ¬     3° structure RNA
                                                                 CºG  ¬           Stem Loop
                                                                 CºG  ¬             structure
                                                                 GºC  ¬ 
                                         5'®C•C•C•A-/     \ -OH®3'
 
3'¬GGGT CGGGCGG ATTCA C T C GCCCG A A A A AAAA C T T GTTTT  ¬5' DNA double strand
            |    |   |    |    |    |    |   |    |   |    |   |    |   |    |   |     |    |    |    |    |   |    |    |     |    |    |    |    |    |    |   |    |    |    |    |    |    |    |   |       
5'®CCCA GCCCGCCT AAGT G AGCG GGC T T T TT TT T G AA C AA AA ®3' 
RNA splicing in eukaryotes
                                                                    gene                                                         
    Chromosomal DNA
nuclear  Primary transcript hnRNA
RNA     RNA splicing removes non-coding regions (exons) to generate a continuous mRNA message 3exon.
 ¬RNA Splicing®messenger mRNA
spliced introns cleaves and degrading to ® nucleotides                                                                        to cytosol
 
RNA editing changes the informational content of RNA
 
Prokaryotes                  In prokaryotes mRNA is used for protein synthesis directly, without alteraction.
                 # DNA bp must be equal #RNA bp
                              ¯¬¾¾¾¾¾¾¾­
                              DNA ¾¾¾¾® mRNA ¾¾¾¾® Protein
 
Eukaryotes
                 # DNA bp DID NOT be equal #RNA bp
                              ¯¬¾¾¾¾¾¾¾¾¾­
                              DNA ¾¾¾¾¾¾® mRNA ¾¾® Protein
                              ¯¾¾¾¾¾¾¾¾¾®­
                              Must Be Modified After Transcrption
 
One Gene = Several Proteins
 
• Fewer Genes Needed                                 RNA editing changes the informational content of RNA
 
• Reduced Genome Size
 
• Mechanism for Construction of New Genes
 
Cytidine Deamination
 
Apo-B (liver)  512 kD
                                         ­ Translation
                  5'  CAA  3'
                                         ¯ Cytidine deaminase
                  5'  UAA  3'
                                         ¯ Translation
Apo-B (SI)  240 kD
 
Apolipoprotein B proteins transport triacylglycerols and cholesterol.
Two 2 kinds of Apo B are known:  Apo B 100 transports endogenously synthesized lipids
                                                         Apo B  48 transports dietary fat
 
Apo B 48 contains 2152 AA amino acids on N-terminal residues of Apo B-100
How is Apo B 48 produced?
Apo-B m-RNA sequence is changed following transcription . A specific Cytidine is deaminated to Uracyl , which leads to change of codon 2153 CAA from glutamate Glu to stop 2153 code UAA.
 
Other examples: glutamite receptors mitochondrial mRNA
 
RNA splicing
 
RNA splicimg is accomplished with the help of
snRNPs (small nuclear RiboNucleoProtein particles) referred to as spliceosome.
snRNPs consist of small nuclear RNA (snRNA and associated proteins).
snRNA sequences are complementary the the pre-mRNA sequences at the splice sites.
                         Cap 5' splice site ¯¯ snRNP      3' splice site ¯¯ snRNP
    PRE-mRNA 5'  3' AAAAA poly(A)
        Splicesome forms ¯ Lariat         and 3' exon 1 is cleaved ¯ 5' splice site of intron cleaved
5'  3'  5'  3' ¾®5' 3'
                     ­ Splicesome ­                                       ¯     ­ Splicesome ­                  Intron will be ­ degraded
Mature mRNA  5' exon 2 cleaves and splices with ¯ 3' exon 2
                                                  Cap 5'  3' AAAAA poly(A)
 
Splicing Signals
 
Consensus sequences for the 5’ and 3’ splice sites of eukaryotic genes contain three 3 important regions.
5’ end of the intron (sequence to be spliced) has a sequence AGGUAAGU
                                                                                 (splicing occurs between the two G nucleotides)
 
3’ end of the intron contains a stretch of >10 or more pyrimidines, followed by NCAGG
                                                                                                           (splicing between two 2 GG nucleotides)
 
Branch point contains an adenosine and is located 20-150 nucleotides upstream from the 3’ splice site.
 
Introns can be 50-10,000 nts long
The rest of the sequence does not affect splicing (ntds changes, deletions), except for rare cases when mutations generate a new consensus sequence resulting in abnormal splicing.
Example: thalassemia is a kind of hereditary anemia that harbors a mutation leading to extra codons in the spliced product - stop codon. Thalassemia syndromes
 
Upstream exon                                              intron                                                      Downstream exon
5' AGGUAAGUA(Py)nNCAGG 3'
                     5' Splice site                          Branch site                          3' Splice site
 
RNA Splicing Mechanism-Step 1
 
2’-OH hydroxyl of the adenosine at the branch site attacks the phospohodiester bond between the upstream exon and the 5’ end of the intron. This leaves the upsteam exon with the 3’ -OH  hydroxy
 
¯UGAAUGGA 5'  Upstream exon
¯                ¯¾® HO¬2’                                  3' Splice site     Downstream exon
¯ A  (Py)nNCAGG 3'
 
Branch A Structure
 
This results in an unusual branched structure with 2’, 5’ phosphodiester bond to A adenosine
 

 
RNA Splicing Mechanism-Step 2
 
2’-OH hydroxyl of the adenosine at the branch site attacks the phospohodiester bond between the
upstream exon and the 5’ end of the intron. This leaves the upsteam exon with the 3’ -OH  hydroxy
 
          ||UGAAUG                                                             3’ GA 5'  Upstream exon
       ||                   O||<--                                                              OH
     ||                  -O-P= O                                                       ||
   ||      Lariat              O    2’                      3' Splice site      ||  Downstream exon
|| A  (Py)nNCAGG 3'
 
RNA Splicing Mechanism-Step 3
 
 
2’-OH hydroxyl of the adenosine at the branch site attacks the phospohodiester bond between the upstream exon and the 5’ end of the intron. This leaves the upsteam exon with the 3’ -OH  hydroxy
 
          ||UGAAUG   Lariat intron  
       ||                   O<--  
     ||                  -O-P= O
   ||  Lariat                  O    2’ 
|| A  (Py)nNCAG-OH
                                           Lariat intron +   spliced product   = exon 1 +  exon 2
                                                                                              5' AGG 3'
 
Splicesome RNA-Protein complexes (U1,U2, U4, U5, U6 snRNPs)
Facilitate RNA Splicing
 
RNA splicing is catalyzed by a spliceosome formed from the assembly of U1, U2, U5 and U4 / U6 snRNPs
 
2’-OH hydroxyl of the adenosine at the branch site attacks the phospohodiester bond between the upstream exon and the 5’ end of the intron. This leaves the upsteam exon with the 3’ -OH  hydroxy
 
U1
||UGAAUGGA 5'  Upstream exon
|| U4 / U6       ||---> HO||<--2’                                  3' Splice site     Downstream exon
|| A  (Py)nNCAGG 3' U4 falls off after catalysis is started.
                                     U2                                                    U5 ­3' Splice site  
 
Splicesome RNA-Protein complexes (U1,U2, U4, U5, U6 snRNPs)
Facilitate RNA Splicing
 
After assembly of spliceosome, the reaction happens in two 2 steps
Step 1 the branch point A nucleotide in the intron sequence attacks 5’ splice site and cleaves it. The two 2 exons ar joined together, and the intron is released as a lariat . Spliceosome complex has a size of 60S, similar to ribosome. This splicing occurs in the nucleus .
         5' SS ||                  bp ||        Py      || 3' SS
5' GUAAG 3'
Pre-mRNA ||<---  + U1
5' GUAAG 3'
                          U1 ||
      U1              ||<--  + U2
G 5'
U2
AAG 3'
                         ||
      U1              ||<--  + U5 + U4 / U6 snRNPs
UG 5'
U2 U4/U6               U5
AAG 3'
                         ||
     U4               ||
UG                         5'
      U2 | U1                U6 | U5
   AAG 3'
                              ||
  U4 |U1     ATP + ||           5'
AG     || 3'
                              ||             
                                         ||->      U4|U1 U2 U6|U5      --->  U4 + U1 + U2 +U6 + U5 dismis splicesome
5'  3' <-||-> AG  --->   +  +  +   +   +  +  +  degradation of Lariat RNA Loop
 
Alternate Splicing
a-Tropomyosine gene
                      DNA
mRNA transcripts
  Striated muscle  -- --------- -- ---------- -- 
 
  Smooth muscle   ---------- -- -------- -------------------
 
  Striated muscle  -- --------- -- ----------  -- -----------
   Myoblast             -- --------- -- ------------- -- -----------
Nonmuscle /
         / fibroblast    -- ------------- -- ------------- -- -----------
   Hepatoma           -- ------------- -- ------------- -- -----------
   Brain                   -- ------------- -- ------------- -- -----------
 
RNA Splicing: RNA Catalysts
 
• Dogma: Only Enzymes (protein catalysts) can catalyze biological reactions.
 
• Crick postulated in the 70’s that RNA may have preceded DNA.
 
• Cech discovered in the 80’s that small RNA’s from Tetrahymena could catalyze self splicing
                                                                without proteins. He called these RIBOZYMES.
 
RNA Self Splicing Mechanism-Step 1
                                                                      ----->
                                                             |—GG||
-----> Upstream Exon                        ||OH<--<-||        P||  ---->  Downstream Exon
5' CUCUCUA----->||       ||  ­        U 3'
                               ||<----AGGGAGG<<--||     ||    ­
                               ||----->----->-------------->----->||
 
RNA Self Splicing Mechanism-Step 2
 
                                                                                   G<-----
®®® Upstream Exon                                   ---> HO-P   ­     Downstream Exon
5' CUCUCU—OH--->||­          ||U||­ 3'
                                ||<---AGGGAGGAG                     ||      ­         ----------------->
                                ||----->----->-------------->------------->||­
 
RNA Self Splicing Mechanism-Step 3
 
----> Upstream Exon ------------------------->  Downstream Exon -->
5' CUCUCU  U 3'   Spliced Product
 
           Lariat Loop ||<--AGGGAGGAG  HO-G Spliced RNA Linear Intron
                               ||----->----->-------------->--------->||­                         
 
Hammerhead Ribozyme
 
                                                                ->A-> 
                                                              U         G
                                                              G          U 
                                                                A       C
                                             5' CG CGG           AGCUCGG  ppp
                                                  |  |   |  |  |             |  |  |  |   |  |  |
                                                 GCGC C          UCGA GCC
                                                              G          C <---Cleavage Site
                                                                AAA=U
    Spliced tertiary 3° structure RNA            C=G  <---Substrate
     hairpin Loop formation Region                 A=U  <---Substrate
                                                                   C       G
   stable hairpin formation                            ->C-> 
 
             Hammerhead Ribozyme

Hammerhead Ribozyme
                      ->A-> 
                  U         G      
                  G          U 
                   A       C      
5' CG CGG           AGCUCGG  ppp
 
                                                   GCGC C                <---  Cleft Site --->                                  UCGA GCC
                                                                 G          C     
                                                                  AAA=U
                                                                       C=G 
       hairpin Loop formation Region                 A=U  Spliced tertiary 3° structure RNA
                                                                     C       G
      stable hairpin formation                           ->C-> 
 
RNA Splicing: Take Home Message
 
1) mRNA can be modified by either deamination or splicing.
 
2) Splicing involves the bringing together of exons (expressed sequences) and the removing of
                                                            introns (intervening sequences) in a very specific and defined way.
 
3) RNA can carry out catalytic enzymatic like reactions.
 
Hammerhead Ribozyme 1RMN.PDB
 
The 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 26-27 is one 1 of these, catalyzing the hydrolysis of an internal phospho-di ester bond. The splicing reaction that occurs in a spliceo-some is believed to rely on a catalytic center formed by the U2, U5, and U6 snRNAs (Fig. 26-16). Also, an RNA component of ribosomes (Protein Metabolism) may participate in the catalysis of protein synthesis.
Exploring catalytic RNAs has provided new insights into catalytic function in general and has important implications for our understanding of the origin and evolution of life on this planet, a topic discussed at the end of the chapter.
 
(b)
 
Figure 26-27. Hammerhead ribozyme.  Certain virus-like elements called virusoids have small RNA genomes and usually require another virus to assist in their replication and/or packaging, Some virusoid RNAs include small segments that promote site-specific RNA cleavage reactions associated with replication.
These segments are called hammerhead ribozymes because their secondary structures 2° are shaped like the head of a hammer. Hammerhead ribozymes have been defined and studied separately from the much larger viral RNAs.
(a) The minimal sequences required for catalysis. The boxed nucleotides are highly conserved and required for catalytic function. The arrow ¯ indicates the site of self-cleavage.
(b) Three-dimensional 3D structure. The strands are colored as magenta and withe. The hammerhead ribozyme is a metallo-enzyme;  Mg2+ ions are required for activity. The phospho-di-ester bond at the site of
self-cleavage is between the two 2 nucleotide residues shown in magenta Cp. PDB file 1RMN.PDB 1994.