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RNA processing (Introduction, Mechanism of processing, RNAediting, Processing of mRNA-5' capping, Polyadenylation, Intron Splicing, Types of splicing, Types of Polyadenylation etc).pptx
This PPT contains, Introduction Mechanisms of RNA processing Processing of pre-mRNA 5’ capping Polyadenylation Types of polyadenylation Intron splicing Exon and intron Definition Splicing apparatus Alternative splicing Trans splicing RNA editing Processing of pre-rRNA Group I and group II introns Processing of pre-tRNA mRNA degradation mRNA surveillance
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Similar to RNA processing (Introduction, Mechanism of processing, RNAediting, Processing of mRNA-5' capping, Polyadenylation, Intron Splicing, Types of splicing, Types of Polyadenylation etc).pptx
RNA processing (Introduction, Mechanism of processing, RNAediting, Processing of mRNA-5' capping, Polyadenylation, Intron Splicing, Types of splicing, Types of Polyadenylation etc).pptx
- 1. Rashmi M G RNAprocessing 1. Introduction 2. Mechanisms of RNA processing 3. Processing of pre-mRNA 4. 5’ capping 5. Polyadenylation 6. Types of polyadenylation 7. Intron splicing 8. Exon and intron Definition 9. Splicing apparatus 10. Alternative splicing 11. Trans splicing 12. RNA editing 13. Processing of pre-rRNA 14. Group I and group II introns 15. Processing of pre-tRNA 16. mRNA degradation 17. mRNA surveillance
- 2. Rashmi M G Source:Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 268
- 3. Rashmi M G Ineukaryotes, • Transcription and translation takes place in different cellular components • Transcription takes place in nucleus; translation happens in cytoplasm • Thus mRNA undergoes processing/ modifications like- cleavage, addition of nucleotides and chemical modification after synthesis In prokaryotes, • Transcription of mRNA and translation occur simultaneously • Thus mRNA undergo little or no modification after synthesis by RNA polymerase Both prokaryotes and eukaryotes modify pre-tRNA, Pre-rRNA Eukaryotes very extensively process pre-mRNA destined to become mRNA Primary transcript of RNA polymerase = pre-RNA Processing if pre-RNA (especially mRNA ) 5’ capping 3’ cleavage/ polyadenylation Splicing RNA editing Pre-RNA Processing Processed RNA Transported to cytoplasm Translated by ribosomes
- 4. Rashmi M G Processingof eukaryotic pre-mRNA 1. 5’ Capping 2. Polyadenylation 3. Splicing 4. RNA editing Processing of eukaryotic pre-rRNA 1. Cutting and end trimming process 2. Chemical modification of rRNA 3. Splicing Processing of pre- tRNA 1. Cleavage 2. Trimming 3. Chemical modification 4. Splicing
- 5. Rashmi M G Processingof eukaryotic pre- mRNA 1. 5’ Capping: • Eukaryotic mRNA consists of enzymatically appended cap structure consisting of 7- methylguanosine residue joined via 5’-5’ triphosphate bridge • Capping of pre-mRNA is catalyzed by enzyme RNA polymerase II Functions of 5’ capping: • Protection of mRNA from degradation • Transport of the mRNA from nucleus to cytoplasm • Binding of ribosome with mRNA Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 272
- 6. Rashmi M G Duringtranscription, 7-methylguanosine is added to the 5’ end of nascent mRNA One subunit of Capping enzyme removes the γ-Phosphate from the 5’ end of the nascent RNA The other subunit transfers the GMP moiety from GTP to the 5’-Diphosphate of the nascent transcript creating the guanosine 5’-5’ triphosphate structure Separate enzymes transfer methyl groups from S-adenosylmethionine to the N7 position of the guanine at the 5’ end of the nascent RNA If mRNA has a methyl group on N7 position of the guanine at the 5’ end, then it is called cap 0 This is the first methylation step and occurs in all eukaryotes Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 386
- 7. Rashmi M G 2.Polyadenylation: • Eukaryotic mRNAs have a series of upto 250 adenosines at their 3’ end called poly (A) tail • A poly (A) tail is added to the 3’ end of the mRNA and plays an important role in mRNA stability, nucleocytoplasmic export and translation • Poly (A) tail is not specified by the DNA and is added to the transcript by a template – independent RNA polymerase called poly (A) polymerase after endonucleolytic cleavage of the nascent RNA near the 3’ terminus • A single processing complex undertakes both cleavage and polyadenylation • A signal is needed for both cleavage and polyadenylation In mammals, signal sequence called poly (A) signal in the mRNA located between 10-30 nucleotides upstream of the polyadenylation site <30 nucleotides downstream of the polyadenylation site is GU rich downstream element Both are binding sites for multi-subunit protein complexes : Cleavage and polyadenylation specificity factor (CPSF) Cleavage stimulation factor (CstF) Generation of poly (A) tail at 3’ terminus first requires cleavage factors: CFI and CFII –acts as endonucleases to cleave the RNA at polyadenylation site- Cleavage step Poly (A) polymerase (PAP) catalyzes the polyadenylation reaction in template independent manner Its substrate is ATP Polyadenylation reaction passes through 2 stages: • Rather short oligo (A) sequence is added to the 3’ end • Oligo (A) tail is extended to the full residue length • This reaction requires another stimulatory factor that recognizes the oligo (A) tail= Poly Adenylate Binding Protein (PABP) which helps the polymerase to add the adenosines, possibly influences the length of the poly (A) tail that synthesized and appears to play a role in the maintenance of the tail after synthesis
- 8. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 387
- 9. Rashmi M G Typesof polyadenylation Alternative polyadenylation • Occurs in mRNA of some protein coding genes • Addition of poly (A) tail at several possible sites • These type of genes have more than one polyadenylation site • Causes the formation of more than one transcript from a single gene • Ex. Alternative polyadenylation occurring during the development of B- lymphocytes Cytoplasmic polyadenylation • Targets mRNA that already contains a short poly (A) tail • Increases the length of the short poly (A) tail before translation • Length of poly (A) tail can be regulated in the cytoplasm • Stored mRNA has a short poly (A) tail • Activation of the mRNA for translation includes lengthening of the poly (A) tail • Catalyzed by a cytoplasmic poly (A) polymerase • Ex. Egg cell stored mRNA in the cytoplasm for later use after the fertilization
- 10. Rashmi M G 3.Intron splicing: • Noncoding DNA is also found within most eukaryotic genes • Such genes have a split structure in which segments of coding sequence (called expressing sequences or exons) are separated by noncoding sequences (intervening sequences or introns) • The entire gene is transcribed to yield a long RNA molecule and the introns are then removed by splicing, so only exons are included in the mRNA • The process of excising the sequences in RNA that corresponds to introns and joining of sequences corresponding to exons is called RNA splicing Types of pre-mRNA introns splicing GU-AG introns splicing AU-AC introns splicing In GU-AG intron, the 1st two nucleotides of the intron sequence are 5’- GU-3’ (5’ splice site or donor site) and the last two 5’-AG-3’ (3’ splice site or acceptor site AU-AC introns are rare class of introns, because the conserved 5’ GU and 3’ AG dinucleotides are replaced by AU and AC respectively Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no. 389
- 11. Rashmi M G Transesterificationreactions: Splicing of GU-AG introns involves 2 transesterification reactions Cleavage of the 5’ splice site occurs by the first transesterification reaction promoted by the hydroxyl group attached to the 2’ carbon of an adenosine nucleotide located within the intron sequence The result of the hydroxyl attack is cleavage of the phosphodiester bond at the 5’ splice site, accompanied by formation of a new 5’-2’ phosphodiester bond linking the first nucleotide of the intron (the G of the 5’-GU-3’ motif) with the internal adenosine This means that the intron has now been looped back on itself to create lariat structure Cleavage of the 3’ splice site and joining of the exons result from a second transesterification reaction, promoted by the 3’-OH group attached to the end of the upstream exon The group attacks the phosphodiester bond at the 3’ splice site, cleaving it and so releasing the intron as the lariat structure, which is subsequently converted back to a linear RNA and degraded At the same time, the 3’ end of the upstream exon joins the newly formed 5’ end of the downstream exon, completing the splicing process
- 12. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 389
- 13. Rashmi M G Exondefinition and Intron Definition: In exon definition: splicing factors recognize the ends of the exons and splice out the introns in between This mechanism is not universal and occurs when the introns are long and the splice sites are weak In intron definition: the ends of introns are recognized In this case, two splice sites are recognized without requiring any sequences outside the intron
- 14. Rashmi M G Splicingapparatus: • The central components of the splicing apparatus for GU-AG introns are the snRNAs called U1,U2,U4,U5 and U6 • These short RNA molecules associate with proteins to form small nuclear ribonucleoproteins (snRNPs) • Cajal bodies within the nucleus are centers of post-transcriptional modifications of snRNAs and snRNPs assembly • snRNPs+ other accessory proteins+ attached to transcript= forms a series of complex, the last complex= Spliceosome • The structure within which the actual splicing reaction occur
- 15. Rashmi M G Processof assembly of snRNPs and various protein factors: Commitment complex (E complex) initiates a splicing activity This complex comprises of: U1-snRNP, which binds to the 5’ splice site by RNA-RNA base pairing (1st step in splicing) Branch point Binding Protein (BBP), which binds branch point, the splicing factor U2AF, binds with the polypyrimidine tract and members of SR protein family SR protein interact with one another via their Arg- Ser-rich regions and also bind to RNA U2-snRNP binds to branch site (E complex is converted to the A complex)
- 16. Rashmi M G Pre-Spliceosomecomplex (A complex) comprises of: Commitment complex + U2snRNP, the latter attached to the branch site At this stage, an association between U1-snRNP and the U2-snRNP brings the 5’ splice site into close proximity to the branch point The Spliceosome is formed when U4/U6-snRNP (a single snRNP containing two snRNAs) and U5-snRNP attach to the pre-Spliceosome complex The other snRNPs involved in splicing associate with the complex in a defined order Pre Spliceosome complex= Commitment complex+ U2-snRNP
- 17. Rashmi M G WhenU4 dissociates from U6-snRNP, U6-snRNA can pair with U2-snRNA to form the catalytic active site (C complex) This results in additional interactions that bring the 3’ splice site close to the 5’ site and the branch point B1 complex formed when, U5 and U4/U6- snRNPs binds to complex This complex is regarded as a Spliceosome, since it contains the components needed for the splicing reaction U1-snRNP released B1 complex is converted into B2 complex The release of U1-snRNP allows U6-snRNP to interact with the 5’ splice site The catalytic reaction is triggered by the release of U4 This requires hydrolysis of ATP Spliceosome
- 18. Rashmi M G Allthree key positions in the intron are now in proximity and the 2nd transesterification occur as a linked reaction, possibly catalyzed by U6-snRNP, completing the splicing process The two exon sequences are, thereby joined to each other and the intron sequence is released as a lariat ATP is required for assembly of the Spliceosome, but the transesterification reactions do not require ATP
- 19. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 360
- 20. Rashmi M G Alternativesplicing • In some cases alternative 5’ and/or 3’ splice sites are used during splicing • This results in the production of more than one mRNA species from a single pre-mRNA • The production of different RNA products from a single product by changes in the usage of splicing junctions is called Alternative splicing • Ex. Drosophila sex determination Trans splicing • In this case exons from two separate RNA transcripts are spliced together to form a mature mRNA molecule • All the products of trans-splicing have the same 5’ exon and different 3’ exons • Ex. Single celled trypanosomes
- 21. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 393
- 22. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 394
- 23. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 464
- 24. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 463
- 25. Rashmi M G 4.RNA editing: • Changing the nucleotide sequence of RNA so that a mature RNA differs from that encoded by the genomic sequence • 2 ways of RNA editing: • Site specific base modification editing (Substitution editing) • Insertion-Deletion editing • Site specific base modification editing (Substitution editing) Ex.1- Deamination 2 common deamination based RNA editing: C →U editing: Found in human mRNA for apolipoprotein B A → I editing Ex.2- Transamination (U →C editing in Wilms Tumor gene) Source: Snustad, Simmons (2012), Principles of genetics, John Wiley& Sons, Inc, Sixth edition, Page no. 273
- 26. Rashmi M G Insertion-Deletion editing • Nucleotides (Uridine containing nucleotides) are inserted into or deleted from specific region of an mRNA after transcription • Editing reactions involve, cleavage, insertions or deletions and ligation • These reactions are catalyzed by the 20S editosome (a protein complex containing all of the enzymatic machinery necessary to catalyze editing) • The sites in the pre-mRNA to be edited are defined by small RNAs that are complementary to edited RNA sequences • These are commonly referred to as guide RNAs (gRNAs) The gRNA has 3 domains: 1. The 5’ region: which is complementary to the substrate pre-mRNA 2. The central domain: which contain the information necessary to insert and/or delete nucleotides in the pre-mRNA to make the edited sequence 3. The 3’ end of the guide RNA: which is characterized by a poly U- tail Depending on the information present in the template region of the gRNA uridylates may either be added to the pre-mRNA by a terminal uridyl transferase alternately uridylates may be deleted by an uridyl exonuclease Following U insertion or deletion the 5’ and 3’ fragments of the pre-mRNA are ligated through the action of editosome RNA ligase Consequences of RNA editing: • Causes changes in function of edited transcripts as compared to the unedited transcripts • Alters codons in mRNAs • May alter splicing pattern by changing splice site • May affect RNA degradation by modifying RNA sequences involved in nuclease recognition • May affect RNA structure dependent activities that involve binding of RNA by proteins
- 27. Rashmi M G Stagesof Editing: gRNA / mRNA duplex formation Cleavage of the mRNA at target site Addition/ deletion of U residue in mRNA Re-ligation of the two fragments of the newly edited mRNA Source: Benjamin A Pierce, Genetics, A conceptual approach, Page no.395
- 28. Rashmi M G Processingof pre-rRNA: In eukaryotes, 1. Cutting and end trimming process • Involves the use of endonucleases (to cut at specific sites within a longer pre-rRNA) and exonuclease (to trim back from the new ends to make the mature product) 2. Chemical modification of rRNA • Requires a class of small RNAs called snoRNAs (Small nucleolar RNAs) • These snoRNAs primarily guide chemical modifications of other RNAs, mainly rRNAs, tRNAs and small nuclear RNAs • 2 main classes of snoRNAs: • C/D box snoRNAs: are associated with methylation • H/ACA box snoRNAs: associated with Pseudouridylation • These snoRNAs base pairs with the rRNA to create the double stranded region that is recognized as a substrate for methylation and Pseudouridylation In prokaryotes, Processing of pre-rRNA involves, 1. Cleavage 2. Trimming 3. Methylation of 30S pre-rRNA
- 29. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no.400
- 30. Rashmi M G GroupI and Group II introns: • These are self splicing introns • Both are found in few prokaryotic genes also Present in few eukaryotic nuclear and organelles genes • They perform splicing without the help of proteinaceous enzymes and acts as ribozymes • These are self splicing RNA molecules that contain their own active site for intron removal and exon ligation • These introns are distinguished by their mechanisms of splicing and unique structures • It was first discovered by T. Cech during studies of splicing mechanism of the group I intron of pre- rRNA in ciliated protozoan Tetrahymena thermophila Group I introns • Are found in the primary transcripts of some nuclear pre- rRNA forming genes and some organelle’s genes • These introns can move • It migrates by DNA-mediated mechanism which is termed as homing Group II introns • Are found in primary transcripts of some organelle’s genes • These introns can move • It migrates by RNA-mediated mechanism • The protein coded by ORF also has reverse transcriptase activity apart from endonuclease activity • The reverse transcriptase generates a DNA copy of the intron using 3’-OH of the double stranded break as a primer for synthesis and the mRNA of the intron as a template • This process is called retrohoming • Both Group I and Group II introns are metalloenzymes • Requires divalent metal cations for activity (usually Mg2+ or Mn2+ ) • Many Group I and Group II introns in organelle genes contain open reading frame (ORF) which codes for a protein called maturase that appears to play a role in splicing and also has endonuclease activity which allows the introns to be mobile
- 31. Rashmi M G Bothclasses of self-splicing introns perform 2 consecutive transesterification reactions in the process of exon ligation Neither class requires ATP for splicing Nucleophilic attack at the 5’-splice site by the 3’-OH of an exogenous (free) guanosine or guanine nucleotide (GMP, GDP or GTP) This reaction adds the G nucleotide or nucleoside onto the 5’ end of the intron and releases the 5’ exon 3’-OH of the 5’ exon that is generated in first transesterification reaction then acts as a nucleophile at the 3’ splice site of the intron This results in precise excision of the intron Ligation of the exons without lariat formation The 2’-OH of a highly conserved A nucleotide (An internal nucleophile) of the intron attacks the 5’ splice site Release of the 5’ exon and formation of lariat structure, whose 5’ end of the intron is covalently attached to the 2’-OH of the bulged A
- 32. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 391
- 33. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 392
- 34. Rashmi M G Processingof pre-tRNA: Involves cleavage > trimming > chemical modification and splicing tRNAs are commonly synthesized as precursor containing extra sequences that are removed by combinations of endonucleolytic and exonucleolytic activities 5’ end of tRNA is generated by a cleavage action catalyzed by the enzyme Ribonuclease P (RNase P), a ribonucleoprotein wherein the RNA component serves as a ribozyme Generation of 3’ end in E coli involves endonuclease RNase E/F and exonuclease RNase D RNase E makes cut at 3’ end then RNase D trims seven nucleotides from this new 3’ end One feature that is common in all tRNAs is the triplet sequence CCA, at the 3’ terminus The triplet sequence is not coded in the genome, but added as a part of tRNA processing by tRNA nucleotidyltransferase
- 35. Rashmi M G Source:Benjamin A Pierce, Genetics, A conceptual approach, Page no. 399
- 36. Rashmi M G mRNAdegradation Prokaryotic mRNA: • The average half life of bacterial mRNAs is only 1.5 minutes • Degradation of mRNA occurs in the 3’ to 5’ direction • Mediated by several endo- and exonuclease • Endonuclease (RNase E and RNase III) make an internal cut in RNA molecules • Exonuclease (RNase II and polynucleotide phosphorylase, PNPase) removes nucleotides sequentially from the 3’ end of an mRNA • In E coli, RNase E and PNPase along with RNA helicase are located within a multiprotein complex called degradosome Eukaryotic mRNA • The average half life of eukaryotic mRNA is 10-20 minutes in lower eukaryotes; to several hours in mammals • Several degradation pathways of mRNA occur in both 5’ to 3’ direction and 3’ to 5’ direction • Cytosolic mRNA are Degraded by 3 different pathways: • Deadenylation dependent pathway: removal of poly (A) tail catalyzed by deadenylase enzyme • Then deadenylated mRNA may either be decapped and degraded by 5’-3’ exonuclease or been degraded by a 3’-5’ exonuclease • Deadenylation independent pathway: mRNAs are decapped and degraded by the 5’-3’ exonuclease • Endonucleolytic pathway: does not involved decapping or Deadenylation
- 37. Rashmi M G 5’-3’degradation pathway (Major) • Deadenylation at the 3’ end triggers decapping at the 5’ end • Decapping reaction occurs by cleavage of 1-2 bases from the 5’ end • Removal of cap triggers the 5’-3’ degradation pathway in which mRNA is degraded rapidly from the 5’ end by the 5’-3’ exonuclease 3’-5’ degradation pathway • Deadenylation of mRNA degraded by the 3’-5’ exonuclease activity • Exosome, which is related to degradosome, degrades the mRNA in the 3’ to 5’ direction • The Exosome is also found in the nucleus, where it degrades unspliced precursors to mRNA
- 38. Rashmi M G mRNAsurveillance: is a conserved mRNA degradation mechanism utilized by organisms to ensure fidelity and quality of mRNA molecules 2 most important mechanisms: Nonsense mediated decay (NMD) • Detection and decay of mRNA transcripts which contain premature termination codons • It checks that mRNA have been properly synthesized and functions Nonstop mediated mRNA decay • Detection and decay of mRNA transcripts which lack in-frame stop codon • These transcripts are identified during translation when the ribosome arrives at the 3’-end of the mRNA and stalls • Exosome degrades the transcript