Year 3 Pharm.D
Dr. V. Chitra
RNA processing
CENTRAL DOGMA
DNA
RNA
PROTEIN
DNA is the genetic material, located in
the nucleus.
mRNA carries a faithful copy of a gene
into the cytoplasm for protein synthesis.
Proteins are enzymatic and structural
elements, made in the cytoplasm.
RNA processing
DNA
discovery of reverse transcriptase (1970)
RNA
RNA can be back-transcribed into DNA
PROTEIN
DNA
pre-mRNA
MATURE
RNA
discovery of pre-mRNA splicing (1977)
PROTEIN
RNA processing
DNA
MATURE
RNA
PROTEIN
PRECURSOR RNAs
Cleavage
Nucleotide addition
Nucleotide insertion
Nucleotide removal
Sequence addition
Sequence removal
Base modification
Sugar modification
pre-rRNA
pre-tRNA
pre-mRNA
Other RNA-related factors affecting expression
abundance (combination of transcription and degradation)
localization
recruitment to ribosomes
RNAs that function in RNA processing
rRNA
snoRNAs
form complexes with protein, direct nt modifications
tRNA
RNase P
snoRNAs
has both RNA and protein components
form complexes with protein, direct nt modifications
mRNA
snRNAs
gRNAs
U1,2,4,5,6 form spliceosomes with many proteins
provide sequence information for RNA editing
miRNAs
siRNAs
important for regulating gene expression
important for regulating gene expression
sno, small nucleolar
sn, small nuclear
RNAs that function in RNA processing
RNA functions in RNA processing
Site of action typically directed by complementary basepairing
Action often catalyzed by associated protein(s)
HOWEVER
some RNAs—ribozymes—have catalytic activity
self-splicing intron in Tetrahymena rRNA
‘hammerhead’ ribozymes self-cleave
snRNAs appear to have catalytic activity in pre-mRNA splicing
rRNA processing
Cleavage:
DNA ETS
Pre-rRNA is cleaved to 18S, 5.8S, 28S rRNAs;
cleavage order is precise (within species).
18S
5.8S
ITS1 ITS2
28S
18S
5.8S
ITS1 ITS2
28S
18S
5.8S
ITS1 ITS2
28S
18S
5.8S
ITS1 ITS2
28S
18S
5.8S
ITS1 ITS2
28S
RNA ETS
ETS
ETS
ETS
ETS
ETS
ETS
ETS
Schematic is
ETS
generic and
not to scale;
tRNA processing
Removal of 5’ leader and 3’ trailer;
order not absolute
CCA may be encoded (prok.) or
added post-transcriptionally (euk.)
Acceptor stem sometimes edited
Some tRNAs have introns in the
anticodon loop
editing
Many nucleotide modifications
intron
rRNA processing in nucleolus
Modification: both bases and sugars are subject to modification
rRNAs ~100 riboses are 2’O-methylated
10 bases methylated
95 Us isomerized to pseudoUs (ψs)
modifications occur prior to assembly into ribosomes
tRNAs ~100 kinds of modified nucleotides
some incorporated during transcription
some chemically modified post-transcription
snoRNAs direct many modifications
RNA modification
snoRNAs
modify rRNAs, tRNAs, miRNAs, siRNAs, and mRNAs
number variable between organisms; more being found
size range ~60 to ~300 nt
encoded individually, in polycistronic clusters, or in introns
Most C/D snoRNAs
(and snRNAs) have a
5’ trimethylguanosine
(TMG) cap. Patients
with motor neuron
degeneration diseases
often develop
antibodies that
recognize TMG caps.
H/ACA snoRNAs
direct pseudouridylation
C/D snoRNAs
direct methylation
mRNA processing
Capping
Splicing
Polyadenylation
Editing
Export
Localization
Translation
Turnover
From birth to death, an mRNA
associates with a variety of
proteins and other RNAs that
modify it directly or affect its
abundance and recruitment to
ribosomes.
Aguilera 2005
mRNP (messenger ribonucleoprotein particle):
mRNA + associated proteins
mRNA processing - capping
5’ capping
required for translation of eukaryotic mRNAs
mediates initial ribosome binding
7-methylguanosine cap added as RNA exits RNApol II
...G linked via a 5’-5’ pyrophosphate bridge
to first nt of mRNA
...G methylated post-addition
...first bases in mRNA may also become methylated
pause after capping, then cap-binding complex (CBC) associates
with cap and elongation proceeds
Aguilar 2005
RNA processing - splicing
• Removes blocks of non-coding sequence (introns: intervening
sequences), ligates the surrounding coding sequences (exons: expressed
sequences).
• Catalyzed by an RNA/protein complex, the spliceosome, which is
composed of five ribonucleoprotein complexes (snRNPs). Each snRNP
has a distinct small nuclear RNA (snRNA): U1, U2, U4, U5, or U6. Each
also has a set of common core proteins and some that are specific to
the particular snRNA.
• Occurs by two transesterification reactions (no energy required)
cis-splicing: both exons on same RNA
trans-splicing: exons on different RNAs