Spreading Knowledge is a great Legacy. MMG

1. Post-transcriptional Modifications of
RNAs in Eukaryotes

2.  Translation closely follows transcription in
prokaryotes
 In eukaryotes, these processes are separated -
transcription in nucleus, translation in cytoplasm

3. mRNA

4. On the way from nucleus to
cytoplasm, the mRNA is
converted from "primary
transcript" to "mature mRNA"

6. Eukaryotic Genes are Split
 Introns intervene between exons
 chicken pro-alpha-2 collagen gene is 40-kbp long, with
51 exons of only 5 kbp total.
 The exons range in size from 45 to 249 bases
 Mechanism by which introns are excised and exons
are spliced together is complex and must be precise

7.  Capping and methylation
 Splicing
 Base modification
 Editing

8. Capping and Methylation
 Primary transcripts (aka pre-mRNAs or
heterogeneous nuclear RNA) are usually first
"capped" by a guanylyl group
 The reaction is catalyzed by guanylyl transferase
 Capping G residue is methylated at 7-position
 Additional methylations occur at 2'-O positions of
next two residues and at 6-amino of the first
adenine

10. 3'-Polyadenylylation
 Termination of transcription occurs only after RNA
polymerase has transcribed past a consensus
AAUAAA sequence - the poly(A)+ addition site
 10-30 nucleotides past this site, a string of 100 to
200 adenine residues are added to the mRNA
transcript - the poly(A)+ tail
 poly(A) polymerase adds these A residues
 Function not known for sure, but poly(A) tail may
govern stability of the mRNA

12. Splicing of Pre-mRNA
Capped, polyadenylated RNA, in the form of a RNP complex, is
the substrate for splicing
 In "splicing", the introns are excised and the exons are
sewn together to form mature mRNA
 Splicing occurs only in the nucleus
 The 5'-end of an intron in higher eukaryotes is always
GU and the 3'-end is always AG
 All introns have a "branch site" 18 to 40 nucleotides
upstream from 3'-splice site
 Branch site is essential to splicing

13. The Branch site and Lariat
 Branch site is usually YNYRAY, where Y = pyrimidine,
R = purine and N is anything
 The "lariat" a covalently closed loop of RNA is
formed by attachment of the 5'-P of the intron's
invariant 5'-G to the 2'-OH at the branch A site
 The exons then join, excising the lariat.
 The lariat is unstable; the 2'-5' phosphodiester is
quickly cleaved and intron is degraded in the nucleus.

15. The Importance of snRNP
 Small nuclear ribonucleoprotein particles - snRNPs,
pronounced "snurps" - are involved in splicing
 An snRNP consists of a small RNA (100-200 bases
long) and about 10 different proteins
 Some of the 10 proteins are general, some are
specific
 snRNPs and pre-mRNA form the spliceosome
 Spliceosome is about the size of ribosomes, and its
assembly requires ATP

17. Assembly of the Spliceosome
 snRNPs U1 and U5 bind at the 5'- and 3'- splice
sites, and U2 snRNP binds at the branch site
 Interaction between the snRNPs brings 5'- and 3'-
splice sites together so lariat can form and exon
ligation can occur
 The transesterification reactions that join the
exons may in fact be catalyzed by "ribozymes"

19. 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

21. Alternative 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

22. 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
 better than having no protein at all

23. rRNA

24.  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

27. tRNA

30.  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
 Followed by splicing a specific segment out of the tRNA
to produce a mature anticodon loop
 Base modification occurs during this process

32. 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

33. RNA editing
 Definition: Any process, other than splicing, that results in a
change in the sequence of an RNA transcript
 Relatively rare
 Discovered in trypanosome mitochondria
 Also occurs in a few chloroplast genes of plants, and at least a few
nuclear genes in mammals.
33

34. RNA editing mechanisms
 Substitution Editing
 Cytidine deaminases
 Adenosine deaminases
 Insertion/Deletion Editing
34

35. Editing by deamination
 ADARs
 Adenosine Deaminases Acting on RNA
 Binds to double stranded RNA
 Convert adenosine to inosine (A-to-I),which the ribosome
translates as a G. Thus a CAG codon (for Gln) can be
converted to a CGG codon (for Arg).
 APOBECs
 Apolipoprotein B mRNA editing Complexes
 Converts cytosine to uracil (C-to-U)
 in RNA
 single-stranded DNA
35

36. Examples of C-to-U editing
 Apolipoprotein B gene (in humans).
 Apo B100 is expressed in the liver
 Apo B48 is expressed in the intestines.
36

37. Insertion/Deletion Editing
 Requires a special class of RNA called guide RNA (gRNA)
 Multiple U’s are inserted into specific region of mRNAs after
transcription (or U’s may be deleted).
 Found in the mitochondria of trypanosomes
 Has also been found to occur with
 RNA transcripts in the mitochondria of the slime mold Physarum
polycephalum.
 In measles virus transcripts
37

38. 38

39. Significance of RNA editing
 It is essential in regulating gene expression of organisms.
 RNA editing mutant was reported with strong defects in
organelle development.
 Deficiency diseases (mostly cancers)
 It is a mechanism to increase the number of different
proteins available without the need to increase the
number of genes in the genome.
 May help protect the genome against some viruses
39

40. Nervous system and RNA editing
 ADARs mediated RNA editing
 In both coding and noncoding transcriptomes
 Editing in non-coding regions (such as microRNA and 3´
UTR) is more frequent than in coding regions
 It is important for avoiding neurological diseases
40

41. 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

42.  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