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O1 rRNA Processing and Ribosome

Section O RNA Processing and RNPs O1 r RNA Processing O2 t RNA Processing O3 m RNA Processing O4 Alternative mRNA Processing. O1 rRNA Processing and Ribosome. Types of RNA processing rRNA processing in prokaryotes rRNA processing in eukaryotes

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O1 rRNA Processing and Ribosome

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  1. Section O RNA Processing and RNPs O1 rRNA Processing O2 tRNA Processing O3mRNA Processing O4 Alternative mRNA Processing Yang Xu, College of Life Sciences

  2. O1 rRNA Processing and Ribosome • Types of RNA processing • rRNA processing in prokaryotes • rRNA processing in eukaryotes • RNPs and their study • Prokaryotic ribosomes • Euokaryotic ribosomes Yang Xu, College of Life Sciences

  3. Types of RNA processing • Definition: RNA processing is the collective term used to describe these alterations to the primary transcript. • Types: The commonest types of alterationsinclude: (i) The removal of nucleotides by both endonucleases and exonucleases; (ii) The addition of nucleotides to the 5'- or 3'-ends of the primary transcripts or their cleavage products; (iii) The modification of certain nucleotides on either the base or the sugar moiety. Yang Xu, College of Life Sciences

  4. P III III P F III III F P F M16 M16 M23 M23 P 16S RNA 5S RNA 23S RNA rRNA processing in prokaryotes Process in E. coli : • There are seven rRNA operons in E. coli, each operon contains one copy of the 5S, 16S and 23S rRNA coding regions, together with some tRNA (1~4); 1. By RNA polymerases, an initial 30S (6000 nt) transcript is made from one of the seven rRNA operons; 2. This 6000 nt transcript then folds and complexes with proteins; 3. 24 specific basemethylations; 4. Cleavage by specific nucleases (RNase III, M5, M16 and M23) to release the mature rRNAs. RNase RNase Yang Xu, College of Life Sciences

  5. 前18S 前5.8S 前28S ETS2 ETS1 47S ITS2 ITS1 RNase 45S 41S 20S 32S 18S 5.8S + 28S rRNA processing in eukaryotes Process in mammals: • rRNA genes are present in a tandemly repeated cluster containing 100 or more copies of the transcription unit (M2); 1. By RNA polymerases I, an initial 47S (13500 nt) transcript is made from one of the units; 2. Cleavages: firstly in the external transcribed spacers (ETSs) 1 and 2; 3.Cleavages: then in the internal transcribed spacer (ITSs); 4. The 5.8S region base-pair to the 28S rRNA before the mature molecules are produced Yang Xu, College of Life Sciences

  6. L1, L2, L3….L31 23S (2900 bases) 5 S (120 bases) S1, S2, S3….S21 16S (1540 bases) 30S Prokaryotic ribosomes The E. coli 70S ribosome is formed from a large 50S and a small 30S sub-unit. The large sub-unit contains 31 different proteins and one each of the 23S and 5S rRNAs. The small sub-unit contains a 16S rRNA molecule and 21 different proteins. r RNA Proteins Sub-units + 50 S + Yang Xu, College of Life Sciences

  7. L1, L2, L3….L45 23S 5.8S (4800+160bases) 5 S (120 bases) S1, S2, S3….S30 80 S 18S (1900 bases) 40S Eukaryotic ribosomes The complete mammalian (rat) 80S ribosome is composed of one large 60S subunit and one small 40S subunit. The 60S subunit contains one 23S rRNA, one 5.8S rRNA, one 5S rRNA and about 45 proteins. The 40S subunit contains an 18S rRNA molecule and about 30 distinct proteins. r RNA Proteins Sub-units + 60 S + Yang Xu, College of Life Sciences

  8. O2 tRNA Processing, RNase P & Ribozymes • tRNA processing in prokaryotes • tRNA processing in eukaryotes • Rnase P • Ribozymes Yang Xu, College of Life Sciences

  9. 1 2 4 3’ A C C 5’ >150 nt 3 tRNA processing in prokaryotes The processing of tRNATyr ofE. coli : • Mature tRNAs are generated by processing longer pre-tRNA transcripts: • Specific exo- and endo-nucleolytic cleavages by RNases D, E, F and P 1. Endo-RNase E or F cleave 3’-end; 2. Exo-RNase D trims the 3'-end to within 2 nt of mature length; 3. Endo-RNase P can then cut to give the mature 5'-end; 4. Exo-RNase D finally removes the two 3'-residues. • Base modifications: which are unique to each particular tRNA type. Yang Xu, College of Life Sciences

  10. 5’ 3’ A A A CA A UG GC UA AU AC U A U C UAC y tRNA processing in eukaryotes ACC 2. tRNA transferase 1. Endo-RNases The processing of tRNATyr ofyeast: • Mature tRNAs are generated by processing longer pre-tRNA with a 16 nt 5’-leader, a 14 nt intron and a 2 nt 3’-end: 1. Specific cleavages by endo-RNases for 16nt 5’-leader and a 2nt 3’-end 2. tRNA transferase adds the sequence 5'-CCA-3' to the 3'-end to generate the mature 3'-end of the tRNA; 3. Removal of the intron by endo-RNases followed by ligation of the half molecules of tRNA; A A CA UA G U 3. Endo-RNases 4.Base modifications: which are unique to each particular tRNA type (see P2). 4. Base modifications Yang Xu, College of Life Sciences

  11. Ribonuclease P (RNase P) • Definition: RNase P is an endo-nuclease composed of one RNA molecule and one protein molecule. It is therefore a very simple RNP. • Structure: In E. coli, RNase P is composed of a 377 nt RNA and a small basic protein of 13.7 kDa. The secondary structure of the RNA has been highly conserved during evolution. • Function: Its role in cells is to generate the mature 5'-end of tRNAs from their precursors. It is a kind of ribozymes, • Location: RNase P are found in both prokaryotes and eukaryotes, being located in the nucleus of the latter where they are small nuclear RNPs (snRNPs). • Reaction condition: The in vitro RNase P ribozyme reaction requires a higher Mg2+ concentration than occurs in vivo, so the protein component probably helps to catalyze the reaction in cells. Yang Xu, College of Life Sciences

  12. Ribozymes Definition:Ribozymes are catalytic RNA molecules that catalyze particular biochemical reactions. Example: 1. RNase P is a common ribozyme that matures tRNA that acts as an endonuclease. 2. Self-splicing intron: There is an intron in the large subunit rRNA of Tetrahymena that can remove itself from the transcript in vitro in the absence of protein (p534). Function of the Protein: The in vitro reaction is about 50 times less efficient than the in vivo reaction, so it is probable that cellular proteins may assist the reaction in vivo. Medical usage: Currently, there is much interest in using ribozymes to inhibit gene expression by cleaving mRNA molecules in vivo, as it may be possible to prevent virus replication, kill cancer cells and discover the function of new genes by inactivating them. Yang Xu, College of Life Sciences

  13. O3 mRNA Processing, hnRNPs & snRNPs • Processing of mRNA • hnRNP • snRNP particles • 5' Capping • 3' Cleavage and polyadenylation • Splicing • Pre-mRNA methylation Yang Xu, College of Life Sciences

  14. Processing of mRNA of prokaryotes • Features: There is little or no processing of mRNA transcripts in prokaryotes. In fact, ribosomes can assemble proteins before mRNA molecules have not yet been completely synthesized. • Prokaryotic mRNA: It is degraded rapidly from the 5’-end and the first cistron (protein-coding region) can therefore only be translated for a limited amount of time. • Stem-loop structures: Some internal cistrons are partially protected by stem-loop structures that form at the 5'- and 3'-ends and provide a temporary barrier to exonucleases and can thus be translated more often before they are eventually degraded. Yang Xu, College of Life Sciences

  15. Processing of the mRNA of Eukaryotes • Pre-mRNAs: Those transcripts that will be processed to give mRNAs are called pre-mRNAs. • Processing: Pre-mRNAs are processed to mature mRNA by: • 5’-capping; • 3’-cleavage and polyadenylation; • splicing and methylation. • hnRNA: Because eukaryotic RNA Pol II transcribes such a wide variety of different genes, the collection of products made by RNA Pol II is called as heterogeneous nuclear RNA (hnRNA). Yang Xu, College of Life Sciences

  16. hnRNP hnRNP:hnRNA + proteins  hnRNP. hnRNA (is mainly pre-mRNA) is synthesized by RNA Pol II. hnRNP Proteins: are classified into protein A~ protein U and thought to help keeping the hnRNA in a single-stranded form and to assist in the various RNA processing reactions. The A, B and C proteins: Each of the three more abundant hnRNP A, B and C proteins has two forms: A1, A2; B1, B2; C1, C2. hnRNP particles: 1 RNA + 9 tetramers  1 hnRNP particle • Purification of this material from nuclei gives a homogeneous preparation of 30-40S particles called hnRNP particles. These particles are about 20 nm in diameter; • Each contains about 600-700 nt of RNA and three copies of three different tetramers: (A1)3B2, (A2)3B2, and (C1)3C2. Yang Xu, College of Life Sciences

  17. Slow Fast hnRNP (Function) Hybridization of RNA molecules in vitro is stimulated by hnRNP proteins: • This finding suggests that hnRNP proteins prevent formation of RNA secondary structures, thereby facilitating base pairing between different complementary molecules. • They may have a similar function in vivo. Yang Xu, College of Life Sciences

  18. snRNP particles snRNP:snRNA + specific proteins  snRNP. snRNAs are mainly synthesized by RNA Pol II. Function:The most abundant snRNPs (U1, U2, U3, U5 and U6) are involved in splicing and methylation in pre-mRNA. Nucleoplasmic snRNPs: The snRNPs contain the 5'-RA(U)nGR-3' sequence and bind eight common proteins in the cytoplasm, become hyper-methylated, and move back into the nucleus. Construction and Work: snRNA snRNA snRNP splicing snRNP Yang Xu, College of Life Sciences

  19. A T C G G G p pp …...OH 3’ ppp 3’ HO 3’HO m7G ppp ……OH 3’ 5' Capping Feature: It is added in reverse polarity(5' to 5') Function: It acts as:  a barrier to 5'-exonuclease attack, but it also promotes  splicing,  transport and  translation. Definition: A 7-methyl-guanosine nucleotide (m7G, or cap) is added to the 5’-end of the transcript when it is about 25 nt long. + PPi mN6A Yang Xu, College of Life Sciences

  20. 5’……AATAAA…(11-20bp)…C A……TTGTGTGTTG…3’ DNA RNA Pol II 5’……AAUAAA…(11-20bp)…C A……UUGUGUGUUG…3’ RNA endonuclease U7-snRNP 5’……AAUAAA…(11-20bp)…C A……UUGUGUGUUG…3’ Poly A transferase Poly A transferase 3' Cleavage and polyadenylation • Function: The poly(A) tail on pre-mRNA is thought to help stabilize the molecule since a poly(A)-binding protein can bind to it, which should act to resist 3'-exonuclease action. • Structureof the 3’-end of mature mRNA: It is generated by cleavage followed by  the addition of a run of A residues which is called the poly(A) tail. UG-rich AAAAA(100-200A)-OH Yang Xu, College of Life Sciences

  21. Intron Exon Exon 5’-end up 3’-end down Intron splicing-I Definition: In eukaryotic pre-mRNA processing, intervening sequences (introns) that interrupt the coding regions (exons) are removed (spliced out), and the two flanking exons are joined. Structure feature: This splicing reaction occurs in the nucleus and requires the intron to have a 5‘-GU, an AG-3’, a poly-pyrimidine tract and a branchpoint sequence (R=A,G; Y=C, U). GU AG ...CURAY …10-40 bp... (U/C)11 branchpoint pyrimidine Yang Xu, College of Life Sciences

  22. 5’ 5’ 5’ 3’ 3’ 3’ AG GU A AG GU A AG GU A Intron splicing-II U1 U2 Function of snRNPs: The splicing reaction is catalyzed by the Ul, U2, U4, U5, U6 snRNPs. 1. The U1 snRNP binds to the 5'-splice site GU, and the U2 snRNP binds to the branchpoint A; 2. The U4, U5 and U6 can then bind, and the intron is looped out and the 5'- and 3'-exons close together. 3. The complex of snRNPs and pre-mRNA is called a spliceosome. 4. After the spliceosome forms, the two-step splicing reaction can occur with release of the intron as a lariat. U5 U4 U6 U5 U4 U6 Spliceosome Yang Xu, College of Life Sciences

  23. 外显子2 Extron2 Extron 2 Extron 1 G G G Intron splicing-III Process: Splicing takes place in a two-step reaction: 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; The second step, cleavage at the 3'-splice site occurs after the G of the AG, as form and is eventually degraded. Yang Xu, College of Life Sciences

  24. Pre-mRNA methylation • Base methylation: Final modification or processing event that many pre-mRNAs undergo is specific methylation of certain bases. • mN6A: In mammalian, 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(R=A,G). • Function: Up to 0.1% of pre-mRNA’s A residues are methylated, and the methylations seem to be largely conserved in the mature mRNA, though their function is unknown. Yang Xu, College of Life Sciences

  25. O4 Alternative mRNA Processing Yang Xu, College of Life Sciences

  26. Alternative splicing • Main ways:The generation of different mature mRNAs from a particular type of gene transcript can occur by varying the use of 5'- and 3'-splice sites (alternative splicing). This can be achieved in four main ways; (i) Using different promoters (ii) Using different poly(A) sites (iii) Retaining certain introns (iv) Retaining or removing certain exons. • Mechanism: Where these events occur differently in different cell types, it is likely that cell type-specific factors are responsible for activating or repressing the use of processing sites near to where they bind. Yang Xu, College of Life Sciences

  27. Exon 1 Exon 3 Exon 4 Exon 1 Exon 2 Exon 3 Exon 4 P1 P2 Exon 2 Exon 3 Exon 4 Alternative splicing • Using different promoters: the choice of promoter (see M4) can forces the pattern of splicing, as happens in the a-amylase and myosin light chain genes. • The exon is transcribed from the upstream promoter has the stronger 5'-splice site which out-competes the downstream one for use of the the first 3'-splice site. Yang Xu, College of Life Sciences

  28. poly(A) site Exon 1 Exon 2 Exon 1 Exon 2 Exon 3 Exon 3 Exon 4 Exon 4 P Exon 1 Exon 2 Alternative splicing • Using different poly(A) sites: In the case of immunoglobulins, use of a downstream poly(A) site includes exons encoding membrane-anchoring regions, and m-Ig is produced; whereas when the upstream site is used these regions are not present and the s-Ig is produced. Yang Xu, College of Life Sciences

  29. Stop codon Exon 1 Exon 2 Exon 3 Exon 1 Exon 2 Exon 3 P Exon 1 Exon 2 Exon 3 Alternative splicing • Retainingcertain introns: In some situations, introns can be retained. If the intron contains a stop codon then a truncated protein will be produced on translation. This can give rise to an inactive protein, as in the case of the P element transposase in Drosophila somatic cells. Yang Xu, College of Life Sciences

  30. Exon 1 Exon 2 Exon 3 Exon 4 Exon 1 Exon 2 Exon 3 Exon 4 P Exon 1 Exon 2 Exon 4 Alternative splicing • Retainingor removingcertain exons: The final type of alternative splicing illustrates that some exons can be retained or removed in different circumstances. • Reason: A likely reason is the existence of a factor in one cell type that either promotes the use of a particular splice site or prevents the use of another. The rat troponin-T pre-mRNA can be differentially spliced in this way. Yang Xu, College of Life Sciences

  31. RNA editing Definition:This is a form of RNA processing in which the nucleotide sequence of the primary transcript is altered by either  changing residues,  deleting residues or  inserting residues at specific points along the molecule. Yang Xu, College of Life Sciences

  32. That’s all for Section O Yang Xu, College of Life Sciences

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