Section O RNA Processing and RNPs O1 rRNA Processing O2 tRNA Processing O3 mRNA Processing O4 Alternative mRNA Processing Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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 Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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 alterations include: (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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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 base methylations; 4. Cleavage by specific nucleases (RNase III, M5, M16 and M23) to release the mature rRNAs. III P F III P F P F RNase RNase M16 M23 P 16S RNA 23S RNA 5S RNA Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

rRNA processing in eukaryotes ETS1 ETS2 ITS1 ITS2 前18S 前5.8S 前28S 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 RNase 45S 41S 20S 32S 18S 5.8S + 28S Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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 23S (2900 bases) L1, L2, L3….L31 5 S (120 bases) + 50 S S1, S2, S3….S21 16S (1540 bases) 30S + Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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 23S 5.8S (4800+160bases) L1, L2, L3….L45 5 S (120 bases) + 60 S 80 S S1, S2, S3….S30 18S (1900 bases) 40S + Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

O2 tRNA Processing, RNase P & Ribozymes tRNA processing in prokaryotes tRNA processing in eukaryotes Rnase P Ribozymes Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

tRNA processing in prokaryotes The processing of tRNATyr of E. 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. 4 2 1 A C >150 nt 5’ 3’ 3 Base modifications: which are unique to each particular tRNA type. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

tRNA processing in eukaryotes The processing of tRNATyr of yeast: 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; ACC 2. tRNA transferase 5’ A 3’ A A C A U G G C U A A U A C U A U C UAC 1. Endo-RNases A A C A U A G U y 3. Endo-RNases 4. Base modifications: which are unique to each particular tRNA type (see P2). 4. Base modifications Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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

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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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). Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

hnRNP (Function) Slow Fast 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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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 snRNP splicing Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

5' Capping 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. 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. p pp …...OH 3’ A T C G ppp G 3’ HO + PPi ppp ……OH 3’ G 3’HO m7G mN6A Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

3' Cleavage and polyadenylation Structure of 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. 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. RNA Pol II DNA 5’……AATAAA…(11-20bp)…C A……TTGTGTGTTG…3’ UG-rich U7-snRNP endonuclease 5’……AAUAAA…(11-20bp)…C A……UUGUGUGUUG…3’ RNA 5’……AAUAAA…(11-20bp)…C A……UUGUGUGUUG…3’ Poly A transferase Poly A transferase AAAAA(100-200A)-OH Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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). Intron Exon GU ...CURAY …10-40 bp... (U/C)11 AG 5’-end up 3’-end down branchpoint pyrimidine Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

Intron splicing-II Function of snRNPs: 5’ 3’ 5’ 3’ 3’ 5’ 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. 5’ 3’ GU AG A U1 U2 5’ 3’ GU AG A U5 U4 U6 3’ AG A 5’ GU U5 U4 U6 Spliceosome Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. 外显子2 G Extron2 G Extron 1 Extron 2 Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

O4 Alternative mRNA Processing Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. Exon 3 Exon 4 Exon 2 P2 Exon 1 P1 Exon 1 Exon 4 Exon 3 Exon 2 Exon 3 Exon 4 Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. Exon 1 Exon 2 P Exon 3 Exon 4 poly(A) site Exon 1 Exon 2 Exon 4 Exon 1 Exon 2 Exon 3 Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

Alternative splicing Retaining certain 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. Exon 2 Exon 3 Exon 1 P Stop codon Exon 1 Exon 3 Exon 2 Exon 1 Exon 2 Exon 3 Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

Alternative splicing Retaining or removing certain 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. Exon 1 Exon 4 Exon 3 Exon 2 Exon 2 Exon 1 P Exon 3 Exon 4 Exon 1 Exon 4 Exon 2 Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

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. Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences

That’s all for Section O Section O: RNA Processing and RNPs. Yang Xu, College of Life Sciences