Download RNA PROCESSING AND RNPs

RNA PROCESSING
AND RNPs
RNA Processing
 Very
few RNA molecules are transcribed
directly into the final mature RNA.
 Most newly transcribed RNA molecules
(primary transcripts) undergo various
alterations to yield the mature product
 RNA
processing is the collective
term used to describe the molecular
events allowing the primary
transcripts to become the mature
RNA.
Cytoplasm
Nucleus or Nucleolus
primary transcript
RNA processing
Romoval of nucleotides
addition of nucleotides
to the 5’- or 3’- ends
modification of certain
nucleotides
mature RNA.
(1) Removal of nucleotides by both
endonucleases and exonucleases
 endonucleases
to cut at specific
sites within a precursor RNA
 exonucleases to trim the ends of a
precursor RNA
 This general process is seen in
prokaryotes and eukaryotes for all
types of RNA
(2) Addition of nucleotides to 5’-or 3’ends of the primary transcripts or
their cleavage products.
Add a cap and a poly(A) tail to pre-mRNA
(3) Modification of certain
nucleotides on either the base or
the sugar moiety.
–Add a methyl group to 2’-OH of ribose in
mRNA (A) and rRNA
–Extensive changes of bases in tRNA
RNPs
Ribonucleoproteins =
RNA protein complexs
 The
RNA molecules in cells usually
exist complexed with proteins
 specific proteins attach to specific
RNAs
 Ribosomes are the largest and most
complex RNPs
3-D structure
Digital cryo-electron micrography
RNP
Ribosomes
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Protein biosynthetic
machinery
Made of 2 subunits
(bacterial 30S and 50S,
Eukaryotes 40S and 60S)
Intact ribosome referred
to as 70S ribosome in
Prokaryotes and 80S
ribosome in Eukaryotes
In bacteria, 20,000
ribosomes per cell, 25%
of cell's mass.
Mass of ribosomes is
roughly 2/3 RNA
Prokaryotic Ribosome Structure
Eukaryotic Ribosome Structure

larger and more complex than
prokaryotic ribosomes, but with similar
structural and functional properties
tRNA PROCESSING, RNASE P
RIBOZYMES
AND
 tRNA processing in prokaryotes
 tRNA processing in eukaryotes
 RNase P
 Ribozymes
tRNA 3-D structure
tRNA processing in prokaryotes
Mature tRNAs are generated by
processing longer pre-tRNA
transcripts, which involves
1. specific exo- and endonucleolytic
cleavage by RNases D, E, F and P
(general) followed by
2. base modifications which are
unique to each particular tRNA
type.
tRNA processing in prokaryotes
Primary transcripts
RNase D,E,F and P
tRNA with mature ends
Base modifications
mature tRNAs
tRNA processing in eukaryotes

1.
2.
3.
The pre-tRNA is synthesized
with a
16 nt 5’-leader,
a 14 nt intron and
two extra 3’-nucleotides.
tRNA processing in eukaryotes
1.
2.
3.
4.
Primary transcripts forms secondary
structures recognized by
endonucleases
5’ leader and 3’ extra nucleotide
removal
tRNA nucleotidyl transferase adds
5’-CCA-3’ to the 3’-end to generate the
mature 3’-end
Intron removal
RNase P

Ribonuclease P (RNase P) is an
enzyme involved in tRNA
processing that removes the 5'
leader sequences from tRNA
precursors
RNase P

RNase P enzymes are found in both
prokaryotes and eukaryotes, being
located in the nucleus of the latter
where they are therefore small
nuclear RNPs (snRNPs)
RNase P
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RNA component can catalyze
pre-tRNA in vitro in the absence
of protein. Thus RNase P RNA is
a catalytic RNA, or ribozyme.
Ribozyme
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Ribozymes are RNAs with catalytic
activity that can catalyze particular
biochemical reactions depending on
their capacity to assume particular
structures
RNase P RNA is a ribozyme.
Ribozymes function during
protein synthesis,
in RNA processing reactions, and
in the regulation of gene expression
Ribozyme

Self-splicing introns: the
intervening RNA that catalyze the
splicing of themselves from their
precursor RNA, and the joining of
the exon sequences
Ribozyme

Self-cleaving RNA encoded by
viral genome to resolve the
concatameric molecules of the
viral genomic RNA produced.
These molecules are able to
fold up in such a way as to
selfcleave themselves into
monomeric.
Ribozyme
Ribozymes can be used as
therapeutic agents in
1. correcting mutant mRNA in human
cells
2. inhibiting unwanted gene
expression
 Kill cancer cells
 Prevent virus replication
The Power of RNA interference
RNA Interference (RNAi) is
able to block selective mRNA
LOSS OF FUNCTION
Easy in yeast
Difficult in mammals
RNAi Pathway
Dicer
RNAi = RNA interference
siRNA = small interfering
RNA
siRNP = small interfering
Ribonucleoprotein
RISC = RNA Induced
Silencing Complex
Inhibition the replication
of SARS virus by RNA
interference
mRNA PROCESSING, hnRNPs
snRNPs
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AND
Processing of mRNA
hnRNP
snRNP particles
5’Capping
3’Cleavage and polyadenylation
Splicing
Pre-mRNA methylation
Processing of mRNA
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There is essentially no processing
of prokaryotic mRNA, it can start
to be translated before it has
finished being transcribed.
Prokaryotic mRNA is degraded
rapidly from the 5’ end
Processing of mRNA in
eukaryotes
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In eukaryotes, mRNA is synthesized
by RNA Pol II as longer precursors
(pre-mRNA), the population of
different RNA Pol II transcripts are
called heterogeneous nuclear RNA
(hnRNA).
Among hnRNA, those processed to
give mature mRNAs are called premRNAs
Processing of mRNA in
eukaryotes
Pre-mRNA
molecules are processed to
mature mRNAs by 5’-capping, 3’cleavage and polyadenylation, splicing
and methylation.
Eukaryotic mRNA processing: overview
hnRNP: hnRNA + proteins
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The hnRNA synthesized by RNA Pol
II is mainly pre-mRNA and rapidly
becomes covered in proteins to
form heterogeneous nuclear
ribonucleoprotein(hnRNP)
The hnRNP proteins are though to
help keep the hnRNA in a singlestranded form and to assist in the
various RNA processing reactions
snRNP particles: snRNA +
proteins
1.
2.
3.
snRNAs are rich in the base uracil,
which complex with specific proteins
to form snRNPs.
The most abundant snRNP are
involved in pre-mRNA splicing,
U1,U2,U4,U5 and U6.
A large number of snRNP define
methylation sites in pre-rRNA.
snRNP particles
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They are synthesized in the nucleus by
RNA Pol II and have a normal 5’-cap.
They are exported to the cytoplasm
where they associate with the common
core proteins and with other specific
proteins.
Their 5’-cap gains two methyl groups
and they are then imported back into
the nucleus where they function in
splicing.
Splicing
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Introns: non-coding sequences
Exons: coding sequences
RNA splicing: removal of introns and joining of exons
Splicing mechanism must be precise to maintain open reading frame
Catalyzed by spliceosome (RNA + protein)
5’ Capping
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Very soon after RNA Pol II starts
making a transcript, and before the
RNA chain is more than 20 -30 nt long,
the 5’-end is chemically modified.
7-methylguanosine is covalently to the
5´ end of pre-mRNA.
Linked 5´  5´
Occurs shortly after initiation
7-methylguanosine (m7G)
Function of 5´ cap
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Protection from degradation
Increasing translational efficiency
Transport to cytoplasm
Splicing of first exon
3’ Cleavage and polyadenylation
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In most pre-mRNAs, the mature
3’-end of the molecule is generated
by cleavage followed by the addition
of a run, or tail, of A residues which
is called the poly(A) tail.
3’ Cleavage and polyadenylation
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RNA polymerase II does not usually
terminate at distinct site
Pre-mRNA is cleaved ~20
nucleotides downstream of
polyadenylation signal (AAUAAA)
~250 AMPs are then added to the 3´
end
Almost all mRNAs have poly(A) tail
Function of poly(A) tail
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Increasing mRNA stability
Increasing translational efficiency
Splicing of last intron
Splicing
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the process of cutting the pre-mRNA
to remove the introns and joining
together of the exons is called splicing.
it takes place in the nucleus before the
mature mRNA can be exported to the
cytoplasm.
Splicing

Splicing requires a set of specific
sequences to be present. The 5’-end
of almost all introns has the
sequence 5’-GU-3’ and the 3’-end is
usually 5’-AG-3’. The AG at the 3’end is preceded by a pyrimidine-rich
sequence called the polypyrimidine
tract
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Sequences of (a) a typical polyadenylation site and (b) the
splice site consensus.
Spliceosome
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Catalyzes pre-mRNA splicing in
nucleus
Composed of five small nuclear RNAs
(snRNAs) and associated proteins
(snRNPs) assembled on the premRNA
Splicing reaction is catalyzed by RNA
Splicing
cycle
Splicing
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Introns: non-coding sequences
Exons: coding sequences
RNA splicing: removal of introns and joining of exons
Splicing mechanism must be precise to maintain open
reading frame
Catalyzed by spliceosome (RNA + protein)
Pre-mRNA methylation
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The final modification or processing
event that many pre-mRNAs
undergo is specific methylation of
certain bases.
The methylations seem to be largely
conserved in the mature mRNA.
ALTERNATIVE
mRNA
PROCESSING
 Alternative processing
 Alternative poly(A) sites
 Alternative splicing
 RNA editing
Alternative processing
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Alternative mRNA processing is the
conversion of pre-mRNA species into more
than one type of mature mRNA.
Types of alternative RNA processing
include alternative (or differential)
splicing and alternative (or
differential) poly(A) processing.
Alternative poly(A) sites
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Some pre-mRNAs contain more
than one poly(A) site and these
may be used under different
circumstances to generate different
mature mRNAs.
In one cell the stronger poly(A) site
is used by default, but in other cell
a factor may prevent stronger site
from being used.
Alternative splicing

(i)
(ii)
(iii)
(iv)
The generation of different mature
mRNAs from a particular type of gene
transcript can occur by varying the
use of 5’- and 3’- splice sites in four
ways:
By using different promoters
By using different poly(A) sites
By retaining certain introns
By retaining or removing certain
exons
Alternative splicing
1. By using different promoters.2. By using
different poly(A) sites.3 By retaining certain
introns.4. By retaining or removing certain
exons
Sex in Drosophila
is largely
determined by
alternative splicing
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Alternative splicing
• the potential for an increase in phenotypic
diversity without increasing the overall number
of genes. Is achieved by altering the pattern of
exons that are spliced together,
• different proteins can arise from the processed
mRNA from a single gene.
Alternative splicing
• Alternative splicing can occur either at
specific developmental stages or in different
cell types.
• the calcitonin gene yields an mRNA that
synthesizes calcitonin (thyroid) or calcitonin
gene– related peptide (CGRP, brain): 2
proteins with distinctly different functions.
• the α-tropomyosin mRNA have at least 8
different alternatively spliced α-tropomyosin
mRNAs.
Alternative splicing
Many defects in the β-globin genes are
known to exist leading to β-thalassemias.
Some of these defects are caused by
mutations in the sequences of the mRNA
required for intron recognition and,
therefore, result in abnormal processing of
the β-globin primary transcript.
RNA editing
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AN unusual form of RNA processing in
which the sequence of the primary
transcript is altered is called RNA
editing.
Changing RNA sequence (after
transcription)
Two types
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
Base modification (A or C deamination)
Base (U) insertion and deletion
End Of The
lecture
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