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O1 rRNA processing and ribosomes O2 tRNA processing, RNase P and ribozymes

Section O—RNA processing and RNPs. Molecular Biology. O1 rRNA processing and ribosomes O2 tRNA processing, RNase P and ribozymes O3 mRNA processing, hnRNPs and snRNPs O4 Alternative mRNA processing. Molecular Biology. O1 rRNA processing and rebosomes. Type of RNA processing

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O1 rRNA processing and ribosomes O2 tRNA processing, RNase P and ribozymes

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  1. Section O—RNA processing and RNPs Molecular Biology • O1 rRNA processing and ribosomes • O2 tRNA processing, RNase P and ribozymes • O3 mRNA processing, hnRNPs and snRNPs • O4 Alternative mRNA processing

  2. Molecular Biology O1 rRNA processing and rebosomes • Type of RNA processing • Most newly transcribed RNA molecule undergo various alterations to yield the mature product. RNA processing is the collective term used to describe these alterations to the primary transcrip. The commonest types of alterations include: • the removal of nucleotides by both endonuckeases and exonucleases； • the addition of nucleotides to the 5’- or 3’- ends of the primary transcrips or their cleavage products； • the modification of certain nucleotides on either the base or the sugar moiety.

  3. Molecular Biology rRNA processing in prokaryotes RNase III RNase III

  4. Molecular Biology rRNA processing in eukaryotes-1 rRNA in eukaryotes is also generated from a single, long precersor meolecule by specific modification and cleavage steps, although these are not so wll understood. In many eukaryotes, the rRNA genes are present in a tandemly repeated cluster containing 100 or more copies fo the transcription unit .it contains one copy of the 18S coding region and one copy each of the 5.8S and 28S coding regions.

  5. Molecular Biology rRNA processing in eukaryotes-2 47S pre-rRNA 45S pre-rRNA ETS：external transcribed spacers ITS：internal transcribed spacers 41S pre-rRNA 32S pre-rRNA 20S pre-rRNA

  6. Molecular Biology rRNA processing in eukaryotes-3 Methylation takes place at over 100 sites to give 2’-O-methylribose and this isnow known to be carried out by a subset of small nuclear RNP particles which are aboundant in the necleolus.

  7. Molecular Biology RNPs and their study • The RNA molecules in cells usually exist complexed with proteins, specific proteins attching to specific RNAs. These RNA-protein complexes are called ribonucleoproteins (RNPs). Several methods are used to study RNPs, including: • Dissociation, where the RNP is purified and separated into its RNA and protein components which are then characterized. • Re-assembly, used to dicover the order in which the components fit togather and, if the components can be modified, it is possible to gain clues as to their individual functions. • Electron microscopy, can allow direct visualization if the RNPs are large enough. • Antibodies to RNPs or their individual components can be used for purification, inhibition of function and, in combination with electron microscopy. • RNA binding experiment can allow whether a particular protein binds to an RNA • RNase proection experiment cam show which [arts of the RNA are protected by bound protein. • Cross-linked experiments using UV light with or without chemical cross-linking agents can show which parts of the RNA and protein molecules are in close contact in the complex. • Neutron and X-ray diffraction can ultimately give the complete 3-D structure.

  8. Molecular Biology Components of the Ribosome-1 30S

  9. Molecular Biology Components of the Ribosome-2 Prokaryotes Eukaryotes 50S 60S 2820 kDa 1590 kDa Large Subunit 28S, 5S, 5.8 S rRNAs 23S, 5S rRNAs 50 proteins 31 proteins 30S 40S Small subunit 930 kDa 1400 kDa 16S rRNA 18 S rRNA 21 proteins 33 proteins mRNA Binding proofreading mRNA Binding proofreading In addition there are many initiation, elongation and termination factors

  10. Molecular Biology Ribosome- prokaryotes Thermus thermophilus 70S ribosome crystallized and solved by x-ray crystallography (Yusupov et al., 2001 Noller laboratory) Large Subunit 50S Small Subunit 30S

  11. Molecular Biology Ribosome-eukaryotes 50 proteins 33 proteins

  12. Molecular Biology O2 tRNA processing, RNASE P and ribozymes •Bacteria has 30-40 tRNA for 61 codon and 20 amino acids •Some tRNA for the same amino acid recognizes more than one codon •More than one tRNA for one amino acid •Animals and plants have 50-100 tRNA •Codon and tRNA usage may differ under diffierent differential stages in the same organism tRNA 70-80 nucleotides

  13. Molecular Biology Robert Holley 1965 – Robert Holley (Cornell Univ.) determined the sequence of the 1st biologically significant nucleic acid Ala-tRNA after 7 years of struggling. Nobel prize, 1968.

  14. Endonuclease 5¢ 3¢ A C Exonuclease (RNase D) RNase P C T Processing involves RNA cleavage and base modifications. T mG P RNase P is a ribozyme P mG = Methylguanosine P = Pseudouridine I T = 4-Thiouridine Anticodon I = 2-Isopentenyladenosine Molecular Biology Prokaryotic tRNAProcessing

  15. Molecular Biology Eukaryotic tRNA processing After the two 3’-nucleotides have been cleaved off, the enzyme tRNA nucleotidyltransferase adds the sequence 5’-CCA-3’ to the 3’-end to generate the mature 3’end of the tRNA. The next step is the removal of the intron, which occurs by endonucleolytic cleavage at each end of the intron followed by lagation of the half molecules of tRNA. The introns of yeast pre-tRNAs can be processed in vertebrates and therefore the eukaryotic tRNA processing machinery seems to have been highly conserved during evolution.

  16. Molecular Biology RNASE P • RNaseP is a ribozyme – RNase P generates 5’ end of tRNAs. • ----S. Altman (shared Nobel Prize with T. Cech) showed RNA component is the catalytic part of the complex and that a protein-free preparation will affect the same catalysis at high salt concentration. • The protein component is very basic.

  17. Molecular Biology Ribozymes • Where is the catalytic activity in RNase P? • RNase P is composed of a 375 nucleotide RNA and a • 20 kDa protein. • The protein component will NOT catalyze cleavage on • its own. • The RNA WILL catalyze cleavage by itself !!!! • The protein component aids in the reaction but is not • required for catalysis. • Thus RNA can be an enzyme. • Enzymes composed of RNA are called ribozymes.

  18. Molecular Biology Discovery of ribozymes Before the discovery of ribozymes, onlyproteins were known to have catalytic activity. The first ribozyme was discovered in the early 1980s by Thomas Cech, who was studying RNA splicing (genetics) in the ciliated protozoan Tetrahymena thermophila. This ribozyme was found in the intron of a RNA transcript and removed itself from the transcript. Dr. Thomas R. Cech

  19. + Pre-rRNA Molecular Biology Self-splicing in pre-rRNA in Tetrahymena : T. Cech et al. 1981 Exon 1 Intron 1 Exon 2 Exon 1 Exon 2 Intron 1 Products of splicing were resolved by gel electrophoresis: pre-rRNA + + + + Nuclear extract - + - + GTP - + + - Additional proteins are NOT needed for splicing of this prerRNA! Do need a G nucleotide (GMP,GDP, GTP or Guanosine). Spliced exon Intron circle Intron linear

  20. Molecular Biology O3 mRNA processing,hnRNP and snRNP There is essentially no processing of prokaryotic mRNA; it can start to be translated before it has finished being transcribed. In eukaryotes, snRNP oparticles interact with hnRNP to carry out some of the RNA processing events. Primary transcripts are modified in four ways: 5’-capping, 3’-cleavage and polyadenylation, splicing and methylation. RNA modification in eukaryotes is essential for functions

  21. Molecular Biology Processing of mRNA The product made by RNA Pol II is referred to as heterogeneous nuclear RNA (hnRNA). Those transcripts that will be processed to give mRNAs are called pre-mRNAs. Pre-mRNA molecules are processed to mature mRNA by 5’-capping, 3’-cleavage and polyadenylation, splicing and methylation.

  22. Molecular Biology Roles of hnRNPs hnRNAs (inculding pre-mRNAs) : heterogeneous nuclear RNAs found as primary transcript from DNAs and involved in RNA nuclear export. Proteins bound to hnRNAs are called as hnRNPs involved A-U. The hnRNP are thought to help keep the hnRNA in a single-stranded form and to assist in the various RNA processing reactions.

  23. Molecular Biology snRNP snRNAs : small nuclear RNAs, function in a variety of nuclear processes including the splicing of pre-mRNA snRNAs (90 - 220 nucleotides) and snRNPs have been suggested as possible splicing factors because -They exist in the nucleus in high concentration -Their abundance correlate with transcriptional activity -They are found in association with mRNA precursor J. Steitz and colleagues took an advantage of antibodies naturally found and identified proteins associated with snRNAs and subsequently proved their link to mRNA splicing. Dr. Joan A. Steitz

  24. Molecular Biology Eukaryotic mRNAs are Capped at Their 5’ End Capping occurs after a short section of the pre-mRNA is synthesized. Capping is a co-transcriptional modification. Capping occurs by addition of a GMP nucleotide to the new RNA transcript in the reverse orientation compared with the normal 3’-5’ linkage, givinga 5’-5’ triphosphate bridge. The reaction is carried out by an enzyme called mRNA guanyltransferase and there can be subsequent methylations of the sugars on the first and second transcribed nucleotides.

  25. Molecular Biology Cont. The cap serves as an export signal to move mRNA out of the nucleus, as a ribosome binding site, and as a protective cap to prevent 5’-3’ exonucleolytic degradation of the mRNA.

  26. Molecular Biology Eukaryotic mRNAs are Polyadenylated at Their 3’ Ends The tail is ~ 200 nucleotides long when first added. The polyA tail stabilizes the mRNA and is important for translation initiation.

  27. Molecular Biology 5’-AAUAAA-3’ polyadenylation signal The cleavage and polyadenylation reaction required that specific sequences be present in the DNA and its pre-mRNA transcript.

  28. Molecular Biology Discovery of the ‘Split Genes’ Dr. Phillip A. Sharp Dr. Richard J. Roberts

  29. Molecular Biology Evidence for Introns

  30. Molecular Biology Intron-exon boundaries have short consensus sequences in the intron Splicing also requireds a set of specific sequences to be present. The 5’-end of almost all introns has the sequence 5’-GU-3’ and 3’-end is usually 5’-AG-3’. The AG at the 3’-end is preceded by a pyrimidine-rich sequence called the polypyrimidine tract. About 10-40 residues upstream of the polypyrimidine tract is a sequence called the branchpoint sequence which is 5’-CURAY-3’ in vertebrates.

  31. Molecular Biology mRNA splicing: Two-step First, the bond in front of the G at the 5’-end of the intron at the so-called 5’-splice site is attacked by the 2’-hydroxyl group of the A residue of the branchpoint sequence to creat a tailed circular molecule called a lariat and free exon 1. in the second step, cleavage at 3’-splice site occurs after the G of the AG, as the two exon sequences are joined togather. The intron is released in the lariat from and is eventually degraded.

  32. U1 U2 U2 U1 U2 U2 U2 Molecular Biology mRNA splicing:snRNP mRNA splicing is accomplished through small RNAs (U1, U2, U4, U5 and U6) and associated proteins (snRNPs) which form a complex called the splicesome. First, U1 binds to the 5’-splice site and the U2 binds to the branchpoint. The tri-snRNP compex of U4, U5 and U6 can the bind, and in so doing the intron is looped out and the 5’- and 3’exons are brought into close proximity.

  33. Molecular Biology Pre-mRNA methylation The final modification or processing event that many pre-mRNA undergo is specific methylation of certain bases. In vertebrates, the most common methylation event is on the N6 position of A residues, particularly when these A residues occur in the sequence 5’-RRACX-3’, where X is rarely G. Up to 0.1% of pre-mRNA A residues are methylated, and the methylations seem to be largely conserved in the mature mRNA, though their function is unknown.

  34. Molecular Biology O4 Alternative mRNA Processing It has become clear that in many cases in eukaryotes a particular pre-mRNA species can give rise to more than one type of mRNA. This can occur when certain exons (alternative exons) are removed by splicing and so are not retained in the mature mRNA product. Addtionally, if there are alternative possible poly (A) sites that can be used, different 3’-end can be present in the mature mRNAs. Types of alternative RNA processing include alternative splicing and alternative poly (A) processing.

  35. Molecular Biology Patterns of Alternative Splicing (A) A cassette exon is defined as a regulated exon that is either included in the mRNA or excluded. (B) Alternative 5’ site. (C) Alternative 3’ site. (D) Alternative initiation. (E) Alternative polyadenylation. (F) Intron retention. (G) Mutually exclusive exons. (H) Others

  36. Molecular Biology Tissue-specificity of Alternative Splicing

  37. Molecular Biology RNAediting An unusal form of RNA processing in which the sequence of the primary transcript is altered is called RNA editing. In mammal, editing cuses a single base change form C to U in the apolipoprotein B pre-mRNA, creating a stop codon in the mRNA in intestinal cells. Many cycles of editing eventually produce the mature mRNA which can be translated.

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