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Ribosomal RNA
Transcription, Processing and Modification
rRNA constitute 80% of the RNA in rapidly dividing cell!
A growing mammalian cell must synthesize approximately10 million copies of each type of ribosomal RNA in eachcell generation to construct its 10 million ribosomes.
Eukaryotic ribosomes have four distinct rRNAs:
– Three in large subunit– One in small subunitIn humans– Large subunit contains – 28S, 5.8S and 5S– Small subunit contains – 18S
28S, 18S and 5.8S derived from the pre-rRNA5S 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‘
Endeder 23S RNA (E.coli)
Lafontaine, D.L.J. and Tollervey, D., Nature Mol.Cell
Biol. 2 (2001), 514
200 rRNA
gene copies per haploide
genome,spread out in small clusters on five chromosomes. Transcription and processing takes place in the nuclear structure callednucleolus.
transcribed spacer DNA
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
5'ETS 18SITS1
5.8SITS2
28S 3'ETS 5'ETS 18SITS1
5.8SITS2
3'ETS25S
A0 A1 D1A2
B1 EC2
B2C10 1 2 3 4 5 6
pseudouridylation 2'-O-methylation pseudouridylation 2'-O-methylation
47S 35Scut at 0, 6 cut at A0
45S 33Scut at 1 cut at A1, A2
41S 20S
27SA3
cut at 2
processing B1, B2
36Scut at 3
27SBprocessing
C1,C232S
cut at 4, 57S
processingE
18S 5.8S 28S
18S 25S5.8S
VERTEBRATES YEAST
A3
A
B
C
D
27SA2cut at A3
cut at D1
proteins involvedbelong to the familyof endonucleases,exonucleases,helicases, snoRNPproteins, exportfactors…
Pulse-Chase assays showing rRNA processing in yeast
Processing of Noncoding RNAs in the Nucleus
snoRNPs – small nucleolar ribonucleoproteinparticles
1. Prozessierung von rRNA2. 2‘-O-Methylierung von rRNA3. Pseudouridinylierung
(Ψ) von rRNA
Human: ~ 150 snoRNAs
in nucleolibox C+D snoRNAsbox H+ACA snoRNAs
E.coli: 4 2‘-O-Methylierungen, 10 ΨHuman: 106 2‘-O-Methylierungen, 91 Ψ
Clusters modifizierter Nukleotide
in den aktiven Stellen, aber nicht konserviert.Prokaryonten: keine snoRNA‘s, enzymat. Methylierungen
(Basenmethylierungen
vielhäufiger 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, Gar1pfür Funktion (Gly/Arg
rich
domain: protein
snoRNP
Interaktion)box C+D: Nop58p: Stabilität, Nop56p und Nop1 (Fibrillarin, GAR Domäne oder RGG box): Funktion.
p
p p
TMG
PRNA pol II
ppp
PRNA pol III
PRNA pol II
E2RNA pol II
E1P
p
E3
p
p
E2RNA pol II
E1P E3
p p
En En+1
repeat 1 repeat 2 repeat n
A
B
C
D
Different strategies ofsnoRNA expression
their host genes belong to the 5’TOP family of vertebrate genes
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)
…back to rRNA maturation…
5’ ETS
Hughes, J. M. X. J. Mol. Biol. 259, 645-654 (1996)
Proposed interaction of U3 snoRNA with the pre-rRNA
Smith, C. M. and Steitz, J. A. Cell 89, 669-672 (1997)
There are numerous snoRNAs involved in rRNA processing andmodification
Two peculiarities of pre-rRNA sequence:
Large numbers of methylated nucleotidesPsuedouridine residues
All modifications occur posttranscriptionallyAltered nucleotides at specific positionsClusteredAll altered nucleotides remain in final productSome unaltered nucleotide sections are discardedFunction of altered domains unclear
Examples of modified bases found in RNA
Dihydrouridine Pseudouridine 1-methylguanosine 7-methylguanosin
1-methyladenosine 2-thiocytidine 5-methylcytidine Ribothymine
pseudouridylationsugar methylation
Guide RNA-Mediated Modificationsof Precursor rRNAs
Kiss, T. Cell 109, 145-148 (2002).
snoRNAs act as guides for 2’-O-methylation and pseudouridylation
•The sites of modification depend on complementarity between the snoRNA andrRNA sequences
•snoRNA-directed modification can take place co-transcriptionally, thusfacilitating 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
( )in yeast
( )in yeast
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 believedto play a general role in RNA conformation and stabilization•Modifications tend to be concentrated in functional rRNA regions and to fine-tune ribosome activity and translation•Modifications are absent from regions where ribosomal proteins bind
The Nucleolus: Ribosomeand RNP Factory - III
Pseudoknot domain
CR4-CR5domain
CR7domain
H/ACA domain
H ACA
Template
IH1
Proposed secondary structure of vertebrate telomerase RNP
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 determinedby the length of the telomeres atthe time of your birth.
Telomerase function
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!
