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 YEAST

ITS1 ITS2 ITS1 ITS2 5'ETS 18S 5.8S 28S 3'ETS 5'ETS 18S 5.8S 25S 3'ETS

0 1 2 3 4 5 6 A0 A1 D1 B1 E C1 B2 A2 A3 C2 B 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 A1, A2

27SA2 41S 20S cut at A3 cut at 2 27SA3 processing B1, B2 36S cut at 3 cut at D1 27SB processing C1,C2 32S cut at 4, 5 7S processing E 18S 5.8S 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 A RNA pol II RNA pol III snoRNA expression P P

TMG ppp

RNA pol II P E1 E2 E3

p p

RNA pol II their host genes belong to the P

5’TOP family of vertebrate genes p p p

RNA pol II

P E1 E2 E3 En En+1

p p p repeat 1 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-methylguanosine 7-methylguanosin

1-methyladenosine 2-thiocytidine 5-methylcytidine 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 fine- tune 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 ACA

Pseudoknot domain H/ACA domain Template 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!