Ribosomal RNA
Transcription, Processing and
Modification
rRNA constitute 80% of the RNA in rapidly dividing cell!
A growing mammalian cell must synthesize approximately
10 million copies of each type of ribosomal RNA in each
cell generation to construct its 10 million ribosomes.
Eukaryotic ribosomes have four distinct rRNAs:
– Three in large subunit
– One in small subunit
In humans
– Large subunit contains – 28S, 5.8S and 5S
– Small subunit contains – 18S
28S, 18S and 5.8S derived from the pre-rRNA
5S from separate RNA – transcribed by RNA pol III
RNA POLYMERASE I - TRANSKRIPTS
Lokalisation: Nucleus:
Transkripte: rRNA‘s (außer 5S RNA)
ITS.... internal transcribed spacer; 5.8S RNA: Homolog zum 5‘ Ende
der 23S RNA (E.coli)
Lafontaine, D.L.J. and Tollervey, D., Nature Mol.Cell Biol. 2 (2001), 514
transcribed spacer DNA
200 rRNA gene copies per haploide genome,
spread out in small clusters on five chromosomes.
Transcription and processing takes place in the nuclear structure called
nucleolus.
The Nucleolus: Ribosome and RNP Factory - I
The Nucleolus: Ribosome and RNP Factory - II
rRNAs – synthesizing the precursor
A dark granule at the base of each fibril is a molecule of RNApolymerase I with the newly
synthesized transcript (fine thread) attached to it. At the speed of about 20 nt/s, over a thousand
transcripts can be synthesized in an hour from a single gene.
Processing of Eukaryotic rRNAs
A
C
VERTEBRATES
18S
5'ETS
0
1
ITS1
ITS2
5.8S
2 3
4
YEAST
28S
5
3'ETS
5'ETS
6
B
ITS1
ITS2
18S
5.8S
A0
A1
25S
3'ETS
D1 B1 E C1
A2 A3
C2
B2
D
pseudouridylation 2'-O-methylation
pseudouridylation 2'-O-methylation
47S
35S
cut at 0, 6
cut at A0
45S
33S
cut at 1
cut at 2
cut at A1, A2
41S
20S
cut at A3
27SA2
27SA3
processing
B1, B2
36S
cut at 3
cut at D1
32S
cut at 4, 5
18S
5.8S
27SB
processing
C1,C2
7S
processing
E
28S
18S
5.8S
25S
proteins involved
belong to the family
of endonucleases,
exonucleases,
helicases, snoRNP
proteins, export
factors…
Pulse-Chase assays showing rRNA
processing in yeast
Processing of Noncoding RNAs in the Nucleus
snoRNPs – small nucleolar ribonucleoprotein
particles
1. Prozessierung von rRNA
2. 2‘-O-Methylierung von rRNA
3. Pseudouridinylierung (Ψ) von rRNA
Human: ~ 150 snoRNAs in nucleoli
box C+D snoRNAs
box H+ACA snoRNAs
E.coli: 4 2‘-O-Methylierungen, 10 Ψ
Human: 106 2‘-O-Methylierungen, 91 Ψ
Clusters modifizierter Nukleotide in den aktiven Stellen, aber nicht
konserviert.
Prokaryonten: keine snoRNA‘s, enzymat.
Methylierungen (Basenmethylierungen viel
häufiger als in Eukaryonten).
Struktur der snoRNA-pre-rRNA interaktion ist wichtig für die Funktion.
Evolution: snoRNAs zuerst nur rRNA prozessieren dann Funktion der
Modifizierungen.
snoRNAs come in the cell
in the form of snoRNPs
proteins carrying
enzymatic activity
snoRNP:
box H+ACA: NAP57/Cbf5, Nhp2p, Nop10p
wichtig für die Stabilität, Gar1p
für Funktion (Gly/Arg rich domain: protein snoRNP Interaktion)
box C+D: Nop58p: Stabilität, Nop56p und Nop1 (Fibrillarin, GAR Domäne
oder RGG box): Funktion.
Different strategies of
snoRNA expression
A
RNA pol II
RNA pol III
P
P
TMG
ppp
B
RNA pol II
P
E1
E2
p
E3
p
C
RNA pol II
their host genes belong to the
5’TOP family of vertebrate genes
P
p
p
p
D
RNA pol II
P
E1
E2
p
repeat 1
E3
p
En+1
En
p
repeat 2
repeat n
…back to rRNA maturation…
Transcription – rRNA Processing
Role of small nucleolar RNAs (snoRNAs)
• snoRNPs associate with the rRNA before it is
fully transcribed
• guide snoRNAs participate in nucleotide
modifications (vast majority, also of other
molecules than rRNAs) and pre-rRNA cleavage
reactions (U3, U8, U13, U14, U22, U17, E2, E3,
probably not directly involved in the catalysis)
5’ ETS
Proposed interaction of U3 snoRNA with the pre-rRNA
Hughes, J. M. X. J. Mol. Biol. 259, 645-654 (1996)
There are numerous snoRNAs involved in rRNA processing and
modification
Smith, C. M. and Steitz, J. A. Cell 89, 669-672 (1997)
Two peculiarities of pre-rRNA sequence:
Large numbers of methylated nucleotides
Psuedouridine residues
All modifications occur posttranscriptionally
Altered nucleotides at specific positions
Clustered
All altered nucleotides remain in final product
Some unaltered nucleotide sections are discarded
Function of altered domains unclear
Examples of modified bases found in RNA
Dihydrouridine
Pseudouridine
1-methyladenosine
2-thiocytidine
1-methylguanosine
5-methylcytidine
7-methylguanosin
Ribothymine
sugar methylation
pseudouridylation
Guide RNA-Mediated Modifications
of Precursor rRNAs
snoRNAs act as guides for 2’-O-methylation and pseudouridylation
(
in yeast
)
(
in yeast
)
•The sites of modification depend on complementarity between the snoRNA and
rRNA sequences
•snoRNA-directed modification can take place co-transcriptionally, thus
facilitating folding of the rRNA precursor
•Fibrillarin, a box C/D-associated protein, is most likely a 2’-O-methyl transferase
•Dyskerin/Cbf, a box H/ACA-associated protein, is a pseudouridine synthase
Kiss, T. Cell 109, 145-148 (2002).
How do the guide-snoRNAs guide?
Schematic structure of the guide snoRNAs
~100 of each class
Some unusual guides
•No single modification appears to be important, but globally they are believed
to play a general role in RNA conformation and stabilization
•Modifications tend to be concentrated in functional rRNA regions and to finetune ribosome activity and translation
•Modifications are absent from regions where ribosomal proteins bind
The Nucleolus: Ribosome
and RNP Factory - III
Proposed secondary structure of vertebrate
telomerase RNP
IH1
CR4-CR5
domain
CR7
domain
H
Pseudoknot domain
Template
H/ACA domain
ACA
Telomerase function
Telomerase is a specialized
reverse transcriptase which
provides the active site for
RNA dependent DNA synthesis.
Germ cells contain telomerase.
Somatic cells do not and the
telomeres shorten with age.
Your life span may be determined
by the length of the telomeres at
the time of your birth.
What is rRNA needed for?
To build the ribosomes (structural role)
To help them function (catalytic role)
Ribosomes
Ribosomes - sites of protein synthesis
assembled in the nucleolus
exported into the cytoplasm
a. Free – unbound in the fluid cytoplasm, produce
proteins for use in the cell
b. Bound – attached to the endoplasmic reticulum,
produce proteins for export or for the plasma
membrane
1. E. coli 70S model:
50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins
30S subunit = 16S (1,542 nt) + 20 proteins
2. Mammalian 80S model:
60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) +
50 proteins
40S subunit = 18S (1,900 nt) + 35 proteins
Mammalian ribosome
Synthesis and processing of 5S rRNA
• Coded by a large number of genes outside of the
nucleolus
• Transcribed by RNA polymerase III
• 5’ end remains unchanged
• 3’ end is truncated
• Following synthesis 5S rRNA is transported to
nucleolus
– Incorporated into assembly of ribosomes
Model of the 90S pre-ribosome
30S
50S
The central region of the interface side of the large subunit is largely devoid of protein, and the
nearest section of protein was found to be 18 Å away from the peptide analogue bound at the
peptidyl transferase centre. The region is entirely composed of tightly packed RNA from
domain V of the 23 S rRNA. Since there is no way in which any protein could come close to
this site, the peptidyl transfer must be an RNA-catalysed reaction.
Ribosome is a ribozyme!