1. RNA processing Splicing and the mechanism of splicing of mRNA
Capping and polyadenylation
Alternative processing
Processing of tRNA and rRNA
Ribozymes
RNA degradation

2. The primary transcript
This is an exact complementary copy of the template strand
mRNA modifications create an open reading frame and permit it to be translated
Splicing removes non-functional regions of the primary transcript yielding mature message
Capping and polyadenylation characterize mRNA processing
tRNA modifications include splicing, cleavage of sequences at the 5’ and 3’ end, and base modification
Mature rRNAs are cut out of a preribosomal primary transcript that includes one copy each of 18, 5.8 and 28S rRNA
1.        What is a primary transcript?
1.        What does splicing do?
2.        On what primary transcripts does splicing operate?
3.        How else is an mRNA modified?
4.        How else is a tRNA modified?
5.        What is a rRNA primary transcript called?
6.        How is it processed?

3. Introns
RNA sequence not present in a mature RNA that is flanked by sequence that is present in the mature RNA
Present in higher organisms more often than lower
Very few introns known in bacteria
The higher the organism,
the more likely introns are present
the more frequently they occur in a single gene
the larger they are
Represent the majority of sequence in most human genes
A few genes are intron-less
They are regulated to yield very strong expression to specific signals
Histone genes
No CAP or tail
1.        What is an intron?
1.        In what organisms are introns more abundant?
2.        In what organisms are introns larger?
1.        What characterizes intron-less genes in higher organisms?
2.        What is an example of an intron-less gene?
What else is unique about histone gene transcripts?

4. Initial description of introns
Initially described in Adenovirus and later in the ovalbumin gene of the chicken
The isolated ovalbumin gene was denatured and rehybridized with mRNA from a chicken egg
The hybrids were examined using electron microscopy
D loops formed, representing single stranded regions of genomic DNA not present in the mature message
1.        What is a D-loop?
2.        What do D loops demonstrate?

5. Introns
Four classes differ in their distribution and reaction mechanisms
Group I – nuclear and non-nuclear rRNA, tRNA and mRNA genes
Group II non-nuclear, non-animal mRNA genes
Nuclear mRNA transcripts
ATP, endonuclease dependent tRNA splicing mechanism
1.        How many classes of introns are there?

6. Group I mechanism Catalyzed by RNA of the primary transcript alone
Self splicing intron
Transesterification reaction requires no energy input
Is spontaneous
The reaction is freely reversible
except that the intron, following removal, is degraded
Requires a guanosine cofactor
The guanosine breaks the upstream exon-intron boundary by linking to the 5’ intronic nucleotide
The free upstream 3’ exonic hydroxyl then attacks and displaces the downstream intron-exon boundary, displacing the 3’ intronic hydroxyl
The intron removed is the linear intronic transcript with an extra G attached to the 5’ end
1.        In what types of genes are group I introns found?
2.        What characterizes a self-splicing intron?
3.        What type of reaction is used by self-splicing introns?
4.        What characterizes transesterification reactions?
5.        Although freely reversible, why are introns not put back into primary transcripts by transesterification reactions?
6.        What cofactor is required by group I introns?
7.        How is the free intron modified following a group one splicing reaction?
8.        It the intron linear or circular after removal?

7. Group II mechanism
Like Group I, except rather than a free guanosine using its 3’ hydroxyl, the attacking hydroxyl is the 2’ hydroxyl of an adenine nucleotide within the intron
The 2’ hydroxyl displaces the 3’ hydroxyl of the upstream nucleoside at the exon-intron boundary
This free 3’ hydroxyl then attacks the downstream intron-exon boundary, displacing the 3’ hydroxyl of the intronic nucleotide
The final product is an unusual lariat structure, where the adenine of the intronic sequence is bonded normally AND through its 2’ hydroxyl to the end of the excised intron
Also self splicing
1.        What attacks the intron exon boundary in a group II reaction?
2.        What is the structure of the excised intron?
1.        What is responsible for the processing of most mRNA?

8. Splicing nuclear mRNA I
Splicing requires small nuclear ribonucleoprotein complexes (snRNP’s)
Contain snRNA’s
Necessary for formation of a spliceosome
1.        What is contained by the snRNP?
2.        How do snRNA’s function?

9. Splicing nuclear mRNA II
Exon-intron boundaries recognized by snRNA’s
Consensus sequences within the introns hybridize the snRNA’s
Proteins and other snRNA’s assemble the spliceosome on the transcript
An unpaired A in the 3’ side of the intron attacks the 5’ exon-intron boundary with a 2’ hydroxyl
This forms a lariat structure
The free 3’ hydroxyl of the upstream exon then displaces the downstream junctional nucleotide
Formation of spliceosome requires ATP but not the splicing reactions themselves
1.        What results from the splicing of nuclear mRNA?
2.        Does the splicing mechanism require ATP?

10. Enzyme dependent splicing
Some yeast tRNA’s
Splicing requires enzymatic recognition and cleavage of both exon-intron junctions
This leaves a cyclic phosphate on the upstream exon
Following cleavage,
The 5’ end is activated by a kinase
A kinase transfers phosphate from a nucleotide triphosphate onto a substrate
The cyclic phosphate is cleaved to a 2’ phosphate and then removed
an RNA-RNA ligase reseals the cut ends
The ligation reaction creates a high energy adenine diphosphate bond at the 5’ hydroxyl
The products are the maturing tRNA and a linear intronic RNA
1.        In what cell type are enzyme dependent splicing reactions carried out?
2.        How does a 2’ phosphate result on the 3’ hydroxyl of an exon boundary in yeast tRNA splicing?
3.        How is the phosphodiester linkage established between exons in the above mechanism?

11. Capping
7-methyl G is added to the 5’ end of mRNA in eukaryotes enzymatically during maturation of message in nucleus
The triphosphate on the 5’ end of an mRNA is dephosphorylated to yield a diphosphate
A GTP is hydrolyzed yielding GMP + pyrophosphate as the GMP is added onto the diphosphate
The final triphosphate contains two phosphates from the mRNA and one from the incoming GTP
The G is then methylated at the seven position
The initial two bases of the mRNA may also be methylated
1.        Where does capping occur?
2.        What is capped?
3.        What is the final structure of the cap?
4.        When is the terminal G methylated?

12. Poly A addition
Transcription proceeds beyond the poly A addition sequence (AAUAAA)
Cleavage of the primary transcript occurs downstream of the AAUAAA
The 3’ hydroxyl is a substrate for the enzyme polyadenylate polymerase
This adds a series of A’s onto the 3’ hydroxyl averaging about 200 bases long
1.        Where is the AAUAAA sequence found?
2.        How is a poly A tail added on?
3.        How long are poly A tails?

13. The functional domains of a protein
The function of a protein may be divided into domains
Simple examples are the 5’-3’ exonuclease, 3’-5’ exonuclease and polymerase domains of DNA polymerase I
Some eukaryotic genes may have evolved by switching functional domains into other genes
Evolving domains is easier than evolving a complete protein
Domains are sometimes reflected in exons
For example, the immunoglobulin domain that embeds IgM into the plasma membrane is coded for by a specific exon at the end of the gene
This results in a protein domain at the end of IgM that attaches it to the membrane
The cell can produce an IgM that is free in the serum by not including that exon in the mature message
1.        What is meant by a functional domain of a protein?
2.        How can RNA processing affect the appearance of functional domains in a protein?
3.       

14. Alternative splicing
This is a method for producing alternative messages from one gene
A primary transcript is made
Different splice products are made that are cell type specific
Cell type specific means that one cell, such as an epithelial cell, will make a different form than another cell, even though the gene making the primary transcript is the same
This happens because the snRNP’s or components of the spliceosomes are different in the two cells
1.        What is meant by cell type specific?
2. What advantage is there to alternatively splicing an mRNA?

15. Altenative splicing: Exon skipping
Splice site choices can exclude an entire exon internal to the message
Myosin heavy chain gene expression skips exons during fly development
Exclusion of a splice junction causes exon skipping
One cell recognizes the downstream (3’) splice junction of the next exon in line
So the 5’ donor site is added to the 3’ acceptor site
In another cell, the first downstream site is not recognized and the next 3’ acceptor site is recognized
This skips both the 3’ acceptor site and the 5’ donor site of the skipped exon
1.        What is meant by exon skipping?

16. Alternative splicing: cryptic splice sites
Alternative splicing can also add exons
The alternative exon is within a gene but not normally recognized
Normal mechanisms can be at work to add the exon in a cell type specific manner
Mutations can also destroy splice junction sequences
Without a normal splice site, the cell may choose a sequence that is similar within an intron or exon that is not normally used
This is a cryptic splice site
A cryptic splice site can result in a less than functional protein
But sometimes having a damaged protein is better than having no protein at all
1.        What is the expected result of the use of a cryptic splice site?

17. Alternative cleavage This is at work with IgM expression
At one stage of the immune response, IgM makes a membrane bound form of an IgM antibody
Upon receiving a signal, the cell converts to making the exact same protein, but lacking the carboxyterminal peptide holding it to the membrane
The conversion occurs because cleavage and polyadenylation exclude the last exon of the primary transcript
How is IgM held on the surface of a cell?
4.        What causes release of IgM?
5.        Is this an example of alternative cleavage or alternative processing of IgM mRNA?

18. Post transcriptional processing of tRNA and rRNA
Prokaryotes
Shown above
Eukaryotes
18S, 5.8S and 28 S rRNA is made as one long transcript by RNA polymerase I from a gene
There are multiple copies of these genes and transcription is almost continuously occurring
Processing is enzymatic, cleaving a final product from the large precursor
1.        What is included on a preribosomal RNA?
1.        What is one justification for producing RNA by this mechanism?

19. This requires enzymatic cleavage of sequences on the ends of the primary transcript
RNAse P (a ribozyme) cleaves the 5’ end, and RNAse D the 3’ end
Following RNAse D cleavage, a CCA sequence is enzymatically polymerized onto the 3’ end of the tRNA
This sequence is necessary for the tRNA to accept and bond to its specific amino acid
This is followed by splicing a specific segment out of the tRNA to produce a mature anticodon loop
Base modification occurs during this process
tRNA processing
1.        What cleaves the 5’ end of tRNA during processing? The 3’end?
1.        What is the central difference between RNAse P and RNAse D?
2.        What is the significance of the CCA sequence on the 3’ end?
3.        How is the CCA added?
4.        How is the anticodon loop created?
5.        What other modifications are carried out on maturing tRNA?

20. Ribozymes
These are catalytic RNAs that mainly participate in the cleavage of RNA
All self-splicing mechanisms are examples of ribozymes
They are not true catalysts because they alter their own structure as a result of catalysis
However some group I introns that are excised can continue to catalyze simple transesterification reactions
The may act as free catalytic agents, however, able to cleave RNA in a sequence specific manner
The hammerhead ribozyme can, in theory, be designed and synthesized in a gene machine to degrade any specific RNA sequence
Ribozymes are, though, unstable and subject to degradation by RNAse in vivo
1.        Why aren’t most ribozymes true catalysts?
2.        What type of ribozyme can act as a true catalyst?

21. RNA degradation
The amount of any substance present depends on its rate of synthesis and degradation
RNA (and protein) levels are controlled at the level of degradation as well as synthesis
Less RNA means less resulting protein from translation
Degradation in eukaryotes proceeds by
Endonucleolytic attack on the poly A tail
Decapping
Exonucleolytic from the 5’ end
The rate of degradation is determined by the sequence and structure of the RNA
Exonucleases attack RNA
Exonuclease attack can be inhibited by
Hairpin loops
Poly A tails
1.        How does RNA degradation proceed?
2.        What structural elements can inhibit exonucleases?