RNA processing involves several steps to convert primary transcripts into mature mRNA in eukaryotic cells. These include 5' capping, 3' cleavage and polyadenylation, and RNA splicing. RNA splicing involves two transesterification reactions that remove introns and join exons. Alternative splicing allows a single gene to produce multiple protein variants. Eukaryotic gene expression is regulated by transcriptional activators and repressors that bind cis-regulatory elements like promoters and enhancers. Activators recruit transcriptional machinery while repressors inhibit transcription. Chromatin structure also influences transcription with acetylation associated with active genes.

1. RNA splicing, processing &
editing
Submitted by
Ranjitha H B
P-2082
BTY

2. Processing of eukaryotic pre-mRNA
• Conversion of primary transcript synthesized by RNA
polymerase II to functional mRNA.
• Processing occur in nucleus as nascent mRNA is being
transcribed
 5’ capping
 3’ cleavage/polyadenylation
 RNA splicing
 RNA editing

3. 5̍ ́capppng
• After nascent RNA, reach a
length of 25-30 nucleotides
• 7-methylguanosine added
through unusual 5’,5’-
triphosphate linkage
• Capping enzyme associates
with phosphorylated CTD of
RNA polymerase II

4. Pre mRNA are associated with hnRNP proteins
• Pre- mRNA associates with heterogeneous ribonucleoprotein
particles (hnRNPs)
• Modular structure- RNA binding domain & interacting domain
• RNA binding motifs-
 RRM-RNA recognition motif
 RGG box
 KH motif
• Functions- prevents formation of 2˚ structures
- Makes pre mRNA substrate for processing
- Transport of mRNA

5. 3’ calepvp e pgd
polyadenylation
• All mRNA except histone mRNA
have 3’ poly (A) tail
• Pre-mRNA is cleaved ~20
nucleotides downstream of
polyadenylation signal (upstream-
AAUAAA downstream- GU rich
(or) simply U rich)
• ~200- 250 A are added to 3´ end

6. Splicing via two transesterification
reactions
• Introns are removed & exons are spliced together
• Short transcripts- RNA splicing follows cleavage &
polyadenylation of 3’
• Long transcripts- splicing begins before transcription
completes
• Consensus sequences 5’ GU- AG 3’ rule , sometimes AU-AC,
branch point sequence & polypyrimidine tract

7. Consensus sequences around 5’ and 3’ splice sites in
vertebrate pre-mRNAs
The central region of the intron, which may range from 40 bases to
500 kilobases in length, generally is unnecessary for splicing to occur.

8. RNA-DNA hybridization studies show that introns
are spliced out during pre- mRNA processing

9. Two transesterification reactions that result in splicing of
exons in pre-mRNA
 In the first transesterification reaction,
the 2’ hydroxyl of the branch point A
residue attacks the phosphate at the 5’
end of the intron. This releases the 5’
exon and creates a lariat structure
within the intron
 In the second transesterification
reaction, the 3’ hydroxyl of the
detached 5’ exon attacks the phosphate
at the 3’ end of the intron, resulting in
release of the intron lariat and ligation
of the exons

10. Model of spliceosome-
mediated splicing of pre-
mRNA
 Splicing RNA splicing occurs in
nuclear particles known as
spliceosomes
 Specificity of splicing comes from
the five small snRNP — RNAs
denoted U1, U2, U4, U5, and U6,
which contain sequences
complementary to the splice
junctions

11. • Chain elongation by RNA polymerase II is coupled to the presence
of RNA-procesing factors
 Capping enzyme & RNA splicing polyadenylation factors associates
with phosphorylated CTD
This mechanism may ensure that pre-mRNA is
not synthesized unless the machinery for
processing it is properly positioned
 Excised introns are degraded primarily by exosomes
-multiprotein complexes that contain 11 3’ to 5’ exonucleases as
well as RNA helicases.
 Exosomes also degrade improperly processed pre-mRNA

12. SR proteins contribute to exon definition in long
pre-mRNA
• Average length of exon app. 150 bases, intron app. 3500 bases
• SR proteins
 RNA binding proteins , interacts with exonic splicing enhancers
 Mediates co-operative binding of U1 snRNP to a true splice site and U2
snRNP to a branch point
• Cross-exon recognition complex (complex of SR proteins, snRNPs &
splicing factor-U2AF)

13. Alternative splicing
• most pre-mRNAs contain multiple introns, different mRNAs can be
produced from the same gene by different combinations of 5' and 3' splice
sites
• Joining of exons in varied combinations controls gene expression by
generating multiple mRNAs (and therefore multiple proteins) from the
same pre-mRNA
• About 50% of human genes transcripts (diversity of proteins encoded by
20,000-25,000 gene)
• Can vary in different tissues and in response to extracellular signals
• Eg., sex determination in Drosophila

14. Alternative splicing is the primarily mechanism
for regulating mRNA processing

16. Processing of rRNA & tRNA
• 80% rRNA, 15% tRNA, 2-5% mRNA
• Basic processing is similar in prokaryotic & eukaryotic cells
• rRNA processing-
• Nucleolus- not surrounded by a membrane, is associated with
chromosomal regions that contain the genes for the 5.8S, 18S, and
28S rRNAs
• Ribosomes of higher eukaryotes- 4 types of RNA designated the 5S,
5.8S, 18S, and 28S rRNAs

17. Ribosomal RNA Genes and the
Organization of the Nucleolus
• Transcribed as a single unit within the nucleolus by RNA polymerase I,
yielding a 45S ribosomal precursor RNA
• Transcription of the 5S rRNA, takes place outside the nucleolus in
higher eukaryotes and is catalyzed by RNA polymerase III
• 200 copies of the gene- 5.8S, 18S, & 28S rRNAs, approximately 2000
copies of the gene- 5S rRNA

18. • genes for 5.8S, 18S, and 28S rRNAs are clustered in tandem arrays
on five different human chromosomes (chromosomes 13, 14, 15, 21,
and 22)
• 5S rRNA genes are present in a single tandem array on chromosome 1
• Following each cell division, nucleoli become associated with the
chromosomal regions that contain the 5.85, 185, and 285 rRNA
genes, which are therefore called nucleolar organizing regions
• Size of the nucleolus depends on the metabolic activity of the cell
(granular component)

20. Processing of rRNA takes place within the nucleolus of eukaryotic cells

21. • In addition to cleavage, the processing of pre-rRNA involves
 base modification, addition of methyl groups to specific
bases(10) & ribose residues(100)
 conversion of uridine to pseudouridine(100)
• processing of pre-rRNA requires the action of both proteins
(300) and RNAs (small nucleolar RNAs -snoRNAs)

22. • Individual snoRNPs consist of single snoRNAs associated
with eight to ten proteins
• snoRNPs then assemble on the pre-rRNA to form processing
complexes
• Some snoRNAs are responsible for the cleavages of pre-rRNA
into 185, 5.85, and 285 products
• most abundant nucleolar snoRNA is U3 (200,000 copies per
cell) cleaves pre-rRNA within the 5' external transcribed
spacer sequences
• U8 snoRNA cleaves pre-rRNA to 5.85 and 285rRNAs
• U22 snoRNA cleaves pre-rRNA to 185 rRNA

23. • Most snoRNAs, however, function in rRNA
synthesis as guide RNAs to direct the specific
base modifications of pre-rRNA, including the
methylation of specific ribose residues and the
formation of pseudouridines
• Most of the snoRNAs contain short sequences of
approximately 15 nucleotides that are
complementary to 185 or 285 rRNA-for
modification

24. tRNA processing
• tRNAs in both bacteria and eukaryotes are synthesized as longer
precursor molecules (pre-tRNAs)
• processing of the 5' end of pre-tRNAs involves cleavage by an
enzyme called RNase P (Sidney Altman)
• 3' end of tRNAs is generated by the action of a conventional protein
Rnase
• addition of a CCA terminus at their 3' ends (site of amino acid
attachment)- CCA terminus is encoded in the DNA of some tRNA
genes, but in others it is not.

25. • extensive modification of bases in
tRNA molecules (app. 10% )
• Some pre-tRNAs, as well as pre-
rRNAs in a few organisms, contain
introns that are removed by splicing
i.e., by endonucleases

26. Differences of pre-tRNA splicing
• Splicing of pre-tRNA catalyzed by proteins not by RNAs
• Intron is excised in one step-simultaneous cleavage of both the
intron ends
• GTP/ATP is required

27. Trans-splicing
• Trypanosomes, euglenoids
• mRNAs are constructed by
splicing together separate RNA
molecules
• Carried out by snRNPs
• Also seen in 10-15% of
mRNA of C. elegans
• Y shaped intron is released

28. Self splicing
• RNA can catalyze the removal of their own introns in the
absence of other protein or RNA factors
• Intron acts as a ribozyme
• described by Tom Cech et.al., during studies of the 285 rRNA
of the protozoan Tetrahymena
• self-splicing RNAs in mitochondria, chloroplasts, and bacteria

29. • 2 self splicing RNAs (based on their reaction mechanisms)
 Group I introns (e.g., Tetrahymena pre-rRNA) cleavage at the
5' splice site mediated by a guanosine cofactor. The 3' end of
the free exon then reacts with the 3' splice site to excise the
intron as a linear RNA.
 Group II introns (e.g., some mitochondrial pre-mRNAs)
closely resemble to nuclear premRNA splicing
Indicates active catalytic components of the spliceosome were
RNAs rather than proteins- snRNAs (U2, U6)

32. RNA editing
• Refers to RNA processing events (other than splicing) that
alter the protein-coding sequences of some mRNAs.
• first discovered in mitochondrial mRNAs of trypanosomes
• also described in mitochondrial mRNAs of other organisms,
chloroplast mRNAs of higher plants, and nuclear mRNAs of
some mammalian genes.

33. • 2 TYPES-
 Base modification: A to I, C to U (Deamination)- vertebrates
 Insertion/Deletion: U insertion or deletion
• RNA editing by the deamination of adenosine to inosine is the
most common form of nuclear RNA editing in mammals. This
form of editing plays an important role in the nervous system

34. Editing of Apolipoprotein B mRNA
Tissue-specific editing of Apo-B mRNA thus results in the expression of structrally and
functionally different proteins in liver and intestine. The full-length Apo-BlOO (app.
500kDa) produced by the liver transports lipids in the circulation; Apo-B48(app. 250kDa)
functions in the absorption of dietary lipids by the intestine.
RNA editing by
deamination

35. Transcriptional activators, repressors
& transcription control

36. • Expression of eukaryotic genes is controlled primarily at the
level of initiation of transcription & also regulated during
elongation
• Cis-Acting Regulatory Sequences: Promoters and Enhancers
 Binding sites for regulatory factors, control the expression of
individual adjacent genes
• Enhancers- sequences stimulate transcription from other
promoters as well (SV40), activity depended on neither their
distance nor their orientation with respect to the transcription
initiation site

37. The activity of enhancer is specific for the
promoter of its appropriate target gene.
Specificity is maintained in part by insulators
or barrier elements, which divide chromosomes
into independent domains and prevent
enhancers from acting on promoters located in
an adjacent domain

38. Structure and Function of Transcriptional
Activators
• Consist of 2 independent domains
 DNA-binding domain- recognizes a specific DNA sequence,
 Activation domain- interacts with Mediator or other
components of the transcriptional machinery
• Eukaryotic cells- about 2500 transcription factors
• They contain many distinct types of DNA-binding domains

39. DNA-binding domains

40. Zinc finger domain
• Most common
• Contains repeats of 2 cysteine & 2 histidine residues that bind
zinc ions- common,
• Identified in the polymerase III transcription factor TFIIIA,
polymerase II promoters- Spl
• Steroid hormone receptors (estrogen & testosterone)-
4 cysteine with central zinc
• α helix interacts with major groove of DNA

41. Helix-turn-helix motif
• First recognized in prokaryotes- E. coli catabolite activator
protein (CAP)
• One helix makes most of the contacts with DNA, while the
other helices lie across the complex to stabilize the interaction
• In eukaryotic cells- homeodomain proteins, role in the
regulation of gene expression during embryonic development

42. Leucine zipper & Helix-loop-helix proteins
• Contain DNA-binding domains formed by dimerization of two polypeptide
chains
• leucine zipper contains 4 or 5 Leucine residues spaced at intervals of seven
amino acids
• Dimerization domain held together by hydrophobic interactions between
leucine side chains- Leucine zipper
• Region rich in positively charged amino acids (lysine and arginine) that
binds DNA
• Helix-loop-helix proteins- dimerization domains formed by 2 helical
regions separated by a loop

43. • Combination of distinct protein subunits can form an expanded
array of factors that can differ both in DNA sequence
recognition and in transcription-stimulating activities
• Activation domains- not well characterized
• Acidic activation domains- rich in negatively charged residues
(aspartate and glutamate); others are rich in proline or
glutamine residues

44. Action of transcriptional
activators
2 mechanisms:
1) they interact with Mediator proteins
and general transcription factors to
facilitate the assembly of a
transcription complex and stimulate
transcription
2) they interact with coactivators that
facilitate transcription by modifying
chromatin structure

45. Eukaryotic Repressors
• Binds to specific DNA sequences and inhibit transcription
• interfere with the binding of other transcription factors to
DNA
• compete with activators for binding to specific regulatory
sequences
• interacts with corepressors -modifies chromatin structure
• Binds promoter or enhancer blocks the binding of the activator

46. Action of eukaryotic repressors
Active repressors- contain specific
functional repressor domains that
inhibit transcription via protein-
protein interactions
Eg., Kruppel gene
repression domain of Kriippel is
rich in alanine residues,
whereas other are rich in proline or
acidic residues.

47. Relationship of Chromatin Structure to
Transcription
• DNA of all eukaryotic cells is tightly bound to histones
• Basic structural unit of chromatin is the nucleosome
• Actively transcribed genes- decondensed chromatin
• Tight winding of DNA around the nucleosome core is major
obstacle to transcription, affecting both the ability of
transcription factors to bind DNA and the ability of RNA
polymerase to transcribe through a chromatin template.

48. • Histone acetylation
• Transcriptionally active chromatin
 Core histones (H2A, H2B, H3 and H4) have two domains:
histone fold domain
 amino-terminal tail domain (rich in lysine- modified by
acetylation)
• Histone deacetylases, which remove the acetyl groups from
histone tails- inhibits transcription

50. • Transcriptionally active chromatin- histone H3, including
methylation of lysine-4, phosphorylation of serine-10, acetylation of
lysines 9, 14, 18, and 23, and methylation of arginines 17 and 26
• Deacetylation & methylation of lysines 9, 27, and 36 is associated
with repression and chromatin condensation
• Nucleosome remodeling factor- sliding of histone octamers along
the DNA molecule- accessibility of specific DNA sequences to
transcription factors

51. DNA Methylation
• DNA is methylated specifically at the cytosines
(C) that precede guanines (G) in the DNA chain
(CpG dinucleotides), and this methylation is
correlated with transcriptional repression.
• Methylation commonly seen in transposable
elements
• regulatory role of DNA methylation- as genomic
imprinting, which controls the expression of some
genes involved in the development of mammalian
embryos

52. Regulation of Transcription-MicroRNAs
(miRNAs)

53. X Chromosome
inactivation
Noncoding RNA transcribed from a regulatory
gene, called Xist, localized to the inactive X,
binding & coats- recruitment of a protein
complex that induces methylation of histone
H3 lysine-27 and lysine-9, leading to chromatin
condensation and conversion of most of the
inactive X to heterochromatin.

54. Thank you