PROCESSING OF M RNA IN EUKAYOTES
CAPPING
TRIPHOSPHATASE (TP) IN CAPPING
removes the terminal phosphate from the first nucleotide
GUANYLYLTRANSFERASE (GT) ADDS A GUANINE
MONOPHOSPHATE NUCLEOTIDE
METHYLTRANSFERASE (MT) ADDS A METHYL
GROUP (CH3)
S-adenosylmethionine
CAPS SERVE MULTIPLE FUNCTIONS
1. protect RNAs from decay by exonucleases, which often require 5′-phosphates to
recognize their substrates.
2. serve as a binding site for proteins such as the cap binding complex (CBC) that
mediate subsequent events, including
splicing, polyadenylation, and nuclear export,
First round of translation
POLYADENYLATION
highly conserved six-nucleotide
(hexanucleotide)
sequence AAUAAA
less well conserved U-rich or
GU-rich downstream sequence element
(DSE)
CLEAVAGE AND POLYADENYLATION SPECIFICITY
FACTOR (CPSF)
contains the endonuclease enzyme that executes the cleavage step.
recruits poly(A) polymerase (PAP), using ATP to add a string of A’s onto the 3′-OH of
the mRNA [a poly(A) tail]
WHY POLY(A) POLYMERASE (PAP) IS DIFFERENT
FROM OTHER POLYMERASES?
unlike DNA polymerases and other RNA polymerases, it does not copy a nucleic acid
template
CSTF (CLEAVAGE STIMULATORY FACTOR)
binds the DSE and helps determine the site of cleavage that is bound by Cleavage
Factors I and II (CFI and CFII).
POLY(A) BINDING PROTEIN (PABP)
During poly(A) synthesis, PABP binds to the tail to
1. protect the mRNA from decay by exonucleases
2. promote translation by interacting with the translation machinery
M RNASPLICING BY SPLICEOSOME
PHILIP SHARP & RICHARD ROBERTS (1977)
RIBOZYME
THOMAS CECH (1981)
INTRON WORLD
~ 5% of the genes in yeast (S. cerevisiae) have introns with single intron (mostly): 50
to 1000 nt with an average length of 250 nucleotides.
85% of human genes have at least one intron, and an average human gene has eight
introns and nine exons.
An extreme example is the gene that is mutated in Duchenne muscular dystrophy, which has 78 introns
and 79 exons spread across 2.3 million base pairs.
Human introns vary in length from 50 to 300,000 nt, with an average of 6000 nucleotides, whereas
the average exon length is 300 nucleotides.
The 20-fold-larger average size of introns relative to exons means that introns account for a much
greater fraction of the human genome than exons.
ALTERNATIVE SPLICING Produces protein isoforms and
expand proteome
ALTERNATIVE SPLICING CAN
PRODUCE PROTEIN ISOFORMS
WITH DIFFERENT
FUNCTIONAL DOMAINS
Fibroblast growth factors (FGFR2):
Alternative splicing results in two
protein isoforms that differ in their
extracellular domains.
Because of this difference, each isoform
binds different growth factors.
PROTEIN ISOFORMS CAN BE PRODUCED IN
PARTICULAR CELLS BY CELL TYPE-SPECIFIC
ALTERNATIVE SPLICING
TRANS-SPLICING LINKS EXONS FROM DIFFERENT
TRANSCRIPTS
The transcript that donates this leader segment is called the
spliced leader RNA (SL RNA).
RNA EDITING
RNA EDITING: MOLECULAR PROCESSES THROUGH WHICH NUCLEOTIDE
SEQUENCES IN RNA S ARE CHANGED AFTER TRANSCRIPTION
Many types of RNA, including tRNAs, rRNAs, mRNAs, and snRNAs, are edited
insertion and deletion of nucleotides
base substitution
ADENOSINE-TO-INOSINE
(A-TO-I) EDITING
A-to-I editing is catalyzed by double-
strand RNA-binding enzymes called
adenosine deaminase acting on RNAs
(ADARs)
1. Changing the amino acid sequence of a
protein
2. affecting regulatory elements in RNAs
that function by base pairing to another
RNA or are bound by a protein
For example, conversion of a stable A-U base
pair into a less stable I-U base pair can alter
splicing by affecting base pairing between
snRNAs and pre-mRNAs.
POST-TRANSCRIPTIONAL CHEMICAL More than 170 modifications are
MODIFICATIONS recorded
RNA NUCLEOTIDE MODIFICATION
m6A is the most common modification
Its role is to destabilize the mRNA
Mostly found in last exon or 3'UTR
RNA EXPORT FROM THE NUCLEUS
MRNA S
& SNRNAS (USED FOR SPLICING) ARE
TRANSPORTED TO CYTOPLASM
Adaptor proteins used for the export:
In human cells,
mRNAs are transported out of the nucleus by the TREX (transcription export) complex,
snRNA are transported by PHAX (phosphorylated adaptor for RNA export)
Both TREX and PHAX interact with their RNA cargo during transcription through binding to the cap
binding complex (CBC)
M RNA DECAY
STRUCTURAL VS. REGULATORY MRNAS HALF LIFE
MRNA DECAY TYPICALLY OCCURS IN THE
CYTOPLASM AND REQUIRES TRANSLATION
a deadenylase, a special type
of exonuclease that
Deadenylation is sometimes Two alternative mechanisms specifically cleaves
followed by removal of the 5′ phosphodiester bonds
m7G cap by the decapping between adenosine
enzyme Dcp1/Dcp2 nucleotides one at a time in
the 3′-to-5′ direction
Deadenylation is followed
by digestion by a 3′-to-5′
exonuclease called the
exosome
5′-to-3′ exonuclease scavenger decapping enzyme
(DcpS) catalyzes the hydrolysis of
the 5′ m7G cap, releasing 5′-m7Gp
THE DIFFERENCE BETWEEN MRNA DECAY BETWEEN
EUKARYOTES AND PROKARYOTES
most decay in eukaryotes is initiated by an exonuclease, not an endonuclease
MRNA SURVEILLANCE IN EUKARYOTIC MRNA
DEGRADATION
Scans the mRNA for the nucleotide sequences
that mark the position where translation of the
mRNA should stop
If the surveillance proteins find a termination
codon in the wrong place, then the cap structure
is removed from the 5’ end of the mRNA and the
molecule degraded from this end by an
exonuclease.
RNA I : RNA INTERFERENCE knockdown and silencing
1990 1998 2002
Co-suppression
RNAi
REMARKABLE FEATURES OF RNAI
(1) RNAi is very specific, and only RNAs with perfect complementarity to the dsRNA
are affected;
(2) RNAi is extremely potent, as only a few dsRNA molecules are required per cell to
inhibit expression of the targeted gene, indicating that the process is catalytic;
(3) RNAi can affect cells and tissues that are far removed from the site of
introduction, indicating that there is an RNA transport mechanism;
(4) RNAi affects the progeny of injected animals, indicating that the targeting
information is heritable.
INDIVIDUAL MRNAS IN EUKARYOTES ARE
DEGRADED BY THE DICER PROTEIN
THE ACTION OF DICER INACTIVATES THE VIRUS GENOME, BUT WHAT
IF THE VIRUS GENES HAVE ALREADY BEEN TRANSCRIBED?
A TYPE OF DICER AFFECTS ENDOGENOUS MRNAS
CALLED FOLDBACK RNAS
MISMATCHES MIRNA-MRNA
Mismatches increase the range of sequences that can be
recognized by a single microRNA would be greatly increased
If the mismatches are greater than 4 nucleotides, translation
comes into halt
