6. RNA Processing
Slides do conjunto (29)
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- Describe how the 5’ and 3’ ends of most eukaryotic mRNAs are formed
- Understand the basic mechanisms of splicing and how splicing regulatory proteins can influence how splice sites are selected
- Understand that alternative splicing can increase the coding capacity of genomes
- Solve problems based on your understanding of splicing
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Overall Production of Mature mRNA
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Capping on 5' End of Transcripts
- 3 enzymes act in succession:
- Phosphatase- removes phosphate from original 5' end of mRNA
- Guanyl transferase- adds guanosine base at 5' end
- Methyltransferase- guanosine gets methylated
- Usually phosphodiester backbone is between 5' and 3' carbons but cap causes linkage of 5' to 5' carbons
- Avoids cap getting broken by enzymes that digest 5' to 3' linkages
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Capping on 5' End of Transcripts
- Capping occurs co-transcriptionally
- Newly formed pre-mRNA is capped early
- Capping enzymes bind phosphorylated RNAP II CTD as it transcribes (ride along)
- When transcript emerges from RNAP II (25-30bp) enzymes add the modification, RNAP pauses whilst modifying and will continue when finished
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Poly-A Tail at 3' End of Transcripts
- Cleavage and Polyadenylation
- The 3' end of mRNA is shorter than the original transcript - cleaved to add the polyA tail on (upto 200 adenosine residues)
- Need a consensus sequence for cleavage and polyadenylation events- mainly CA dinucleotide
- CA cleavage site is flanked with other consensus sequences upstream and downstream where proteins can bind
- When cleavage occurs the 3' end is degraded in the nucleus and get addition of polyA
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Poly-A Tail at 3' End of Transcripts
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Proteins Involved in Cleavage/Adding PolyA
- Proteins recognise and bind the flanking sequences:
- Cleavage stimulation factor F (CstF)
- Cleavage and polyadenylation specificity factor (CPSF)
- These proteins also ride along the phosphorylated CTD tail of RNAP II during transcription
- When the sequences they bind are transcribed they transfer to the 3' end sequences on mRNA
- The proteins then bind each other and present CA nucleotide cleavage site to cleavage factors
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Proteins Involved in Cleavage/Adding PolyA
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- An enzyme poly-A-polymerase (PAP) sequentially adds ~200 A nucleotides (from ATP precursor)
- Poly-A binding proteins (PABP) assemble onto the PolyA tail
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Why Add 5' Cap and 3' PolyA Tail?
- Stabilisation:
- Cap, PolyA tail and PolyA binding proteins promote stabilisation
- PolyA proteins bind to other proteins that bind to Cap= circulisation of mRNA which protects ends being chewed by enzymes
- Leave nucleus into cytoplasm which needs stabilised mRNA
- Involvement in export:
- mRNA needs to be transported to cytoplasm to be translated
- Proteins that do shuttling of RNAs out of nucleus recognise cap and polyA/(PABPs)
- Involvement in translation:
- Translation is initiated through cap binding proteins that recognise cap
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- Need to accurately remove introns as single base errors will shift reading frames
- Introns aren't junk:
- Allow alternative splicing
- Promote gene expression
- Promote export out of the nucleus
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RNA Splicing: Sequence Signal for Splice Site
- Signal to guide machinery to point of splicing
- High conservation of sequence is found only immediately within intron at junctions (at exon-intron boundary)
- Generally GU as first base (5' splice site)
- Often run of pyrimidines then AG (3' splice site)
- A= branchpoint site
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- Nucleophilic attack of 2'OH from A onto exon 1 (5' splice site)
- Releases exon 1 and forms loop back on itself
- Nucleophillic attack of 3' OH from exon 1 onto exon 2 (3' splice site)
- Exons join together and excising of lariat structure
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- Performs splicing
- A highly complex molecular machine composed of small nuclear ribonucleoprotein particles (snRNPs/snurps)
- snRNPs made of U RNAs and proteins
- snRNPs find the spice site boundaries to ensure reactions happen in the right place
- 5 U RNAs in total and multiple proteins involved
- Consensus sequences recognised by the RNA component, each snRNP has 1 RNA and lots of proteins
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- U1 snRNP binds 5' splice site and U2 snRNP binds branch point
- RNA pairing is crucial in bringing the spliceosome to the correct place in the pre-mRNA
- U1 snRNA pairs with pre-mRNA at exon intron boundary of 5' splice site
- U2 snRNA pairs at branch point
- Not perfectly paired- binding of U2 to branch point forces A out so it is unpaired and activated for nucleophilic attack on 5' splice site
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- U1 is displaced by U4/6, U6 interacts with U2, U5 binds the 5' exon sequence
- U6 RNA pair with intronic RNA at 5' splice site and with U2 RNA
- Pairing of U2 and U6 ensures A is at the exact right position of the 5' side of GU (5' splice site) for nucelophilic attack onto exon 1
- Two step trans-esterification (2 Nu attacks) and U5 aligns the 5' and 3' exons for ligation
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Challenges of Splice Site Identification
- Most genes have multiple introns- need correct exons to join
- Adv that co-transcriptionally as not all introns out at one time
- Many splice site consensus sequences are not perfectly conserved
- Requires RNA-RNA interactions, weak splice sites have weak interactions between RNA-RNA (less complementary)
- So spliceosome needs more help, there are other sequences in mRNA that can be recognised by RNA binding proteins and help recruit the spliceosome
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Other Sequences that Recruite Spliceosome
- Exonic splicing enhancers (ESEs) interact with SR proteins (conserved family of Serine/Arginine rich splicing factors)
- Single base change in ESE can affect binding of SR proteins, therefore effect whether splicing occurs
- SR proteins recognise sequences within exons (ESEs) and recruit and stabilise splicing machinery via protein-protein interactions
- SR proteins regulated (eg phosphorylation) to activate/deactivate
- Way of achieving alternative splicing
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- Increase protein coding capacity of higher eukaryotes
- But most genes have a major isoform and the proteome is smaller than that predicted from the transcriptome
- Proteome= entire set of proteins expressed in a cell at a certain time
- Transcriptome= entire set of RNAs expressed in a cell at certain time
- Impact of alternative RNA splicing:
- In 5'UTR= altered translation, including removal of regulatory elements that impact translation
- In protein coding sequence (ORF)= altered protein structure, produce different protein isoforms
- In 3'UTR= altered mRNA stability
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Patterns of Alternative Splicing
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Example of Effect of Alternative Splicing
- The primary signal for determining whether fly develops as male or female is X chromosome/autosome ratio:
- Female= 2X chromosomes and 2 sets of autosomes (ratio=1)
- Male= 1X chromosome and 2 sets of autosomes (ratio = 0.5)
- Cascade of alternative splicing events in 3 genes-->
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- Example of exon skipping/inclusion
- SPF45 = splicing enhancer that promotes inclusion of exon 3
- SXL = acting as an inhibitor of exon 3 inclusion
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- Males:
- Sxl transcript includes exon 3 that has premature stop codon
- SPF45 binds AG dinucleotide and promotes exon 3 joining with exon 2
- Because of pre-mature stop codon, produce non-functional sex lethal protein
- Females:
- SXL binds site adjacent SPF45 and interferes with its activity as a splicing enhancer
- Exon 3 is skipped, exon 2 joins with exon 4
- Produce functional sex lethal protein because 3' splice site of exon 3 not used
- SXL regulates alternative splicing of its own pre-mRNA
- Females
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2. Transformer (tra) gene
- Males:
- Proximal 3' splice site used, promoted by U2AF auxiliary factor
- mRNA produced contains premature stop codon= non-functional tra protein
- Females:
- SXL product binds pre-mRNA and shifts the binding of U2AF to a more distal 3' splice site of exon 2 - SXL acts as splice site repressor
- Premature stop codon not included so functional tra protein formed
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2. Transformer (tra) gene
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- Male:
- Dont have transformer product
- 3' splice site immediately upstream of exon 4 not recognised
- Exon 4 skipped, use a more distal polyA site not on exon 4
- Females:
- Tra acts as promoter of exon inclusion by complexing with SR protein (RBP1) and SR-like protein (TRA2) to bind ESEs (sequence enhancers) in exon 4
- Recruit splicing machinery to 3' splice site
- Exon 4 has polyA signal which results in cleavage at that site, exon 5 and 6 not included
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- Both male and females produce variants of DSX protein- male protein represses female differentiation genes
- Both have same N- because have same first 3 exons but have specific C-
- Males have longer version
- Controlled by proteins that recognise and block or recruit splicing activity
