6. RNA Processing

Learning Outcomes: L13

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

Overall Production of Mature mRNA

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Capping on 5' End of Transcripts

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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

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

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

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

Proteins Involved in Cleavage/Adding PolyA

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Following Cleavage

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 

RNA Splicing

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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

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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RNA Splicing: Mechanism

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) Ex​​​​ons join together and excising of lariat structure

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Spliceosome

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

Spliceosome Steps of Splicing

 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 

Spliceosome Steps of Splicing

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Spliceosome Steps of Splicing

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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Spliceosome Steps of Splicing

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 

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 

Alternative Splicing

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

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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1. Sex-lethal (sxl) gene

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

1. Sex-lethal (sxl) gene

​​​​​​​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

3. Doublesex (dsx) Gene

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

Summary of Example

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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